Multi-wavelength phototherapy systems and methods for the treatment of damaged or diseased tissue

ABSTRACT

Provided are multi-wavelength phototherapy systems and methods for the treatment of a disorder or disease, including multi-wavelength low level light therapy (“LLLT”), in particular to multi-wavelength LLLT and other phototherapy systems and methods for improving functionality in and/or restoring functionality to a cell and/or tissue through the coordinated and targeted delivery to the cell or tissue of two or more doses of light having distinct wavelengths, wherein the two or more doses of light, when delivered in a coordinated fashion, can stimulate the activity of two or more light sensitive factors that, when activated, provide and/or enhance a desired target cell functionality. Also provided are multi-wavelength phototherapy systems and methods for treatment of disorders and diseases that are associated with a diminished functionality in a cell of a patient afflicted with the disorder or disease, which can be adapted for therapeutic use by the coordinated and targeted delivery of two or more distinct wavelengths of light to a cell or tissue in a patient afflicted with a disorder and/or disease to restore or enhance, respectively, a diminished functionality to a cell and/or tissue that is associated with the disorder and/or disease healing the disorder or reversing and/or slowing the progression of one or more aspect of the disorder and/or disease. Within certain aspects, provided are multi-wavelength phototherapy systems and methods for the treatment of a damaged or diseased ocular tissue in an eye of a human, including ocular tissue that is associated with dry macular degeneration, which systems and methods include the delivery to the eye of two or more therapeutically effective doses of light, wherein the two or more therapeutically effective doses of light include a first dose of light having a first wavelength and a second dose of light having a second wavelength. These multi-wavelength phototherapy systems and methods can be used advantageously in combination with the administration or delivery of one or more additional therapeutics, including one or more small molecule, biologic, or other therapeutic treatment regimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. non-provisional patent application was filed on Dec. 1, 2015 and claims priority as a continuation-in-part from PCT Patent Application No. PCT/US15/49261, which was filed on Sep. 9, 2015 and which claims the benefit of U.S. Provisional Patent Application Nos. 62/048,182, 62/048,187, and 62/048,211, each of which was filed on Sep. 9, 2014. PCT Patent Application No. PCT/US15/49261 and U.S. Provisional Patent Application Nos. 62/048,182, 62/048,187, and 62/048,211 are incorporated herein by reference in their entirety.

BACKGROUND OF THE DISCLOSURE

1. Technical Field

The present disclosure relates, generally, to multi-wavelength phototherapy, including multi-wavelength photobiomodulation (“PBM”). More specifically, disclosed herein are non-invasive phototherapy systems and methods for improving functionality in and/or restoring functionality to a cell and/or tissue through the coordinated and targeted delivery to the cell or tissue of two or more doses of light having distinct wavelengths, wherein the two or more doses of light, when delivered in a coordinated fashion, can modulate the activity of two or more light sensitive factors or photoacceptors that, when activated, can enhance or inhibit a desired target cell functionality. The present disclosure also concerns the treatment of disorders and/or diseases that are associated with an absence and/or diminished functionality in a cell of a patient afflicted with the disorder and/or disease. The multi-wavelength phototherapy systems and methods disclosed herein can, therefore, be adapted for therapeutic use by the coordinated and targeted delivery of two or more distinct wavelengths of light to a cell or tissue in a patient afflicted with a disorder or disease to enhance a diminished functionality, reduce a hyperactive functionality, or correct an altered functionality in a cell or tissue that is associated with the disorder or disease thereby reducing the symptoms or slowing the progression of one or more aspect of the disorder and/or disease.

2. Description of the Related Art

Light can act on different mechanisms within cellular tissue to stimulate or suppress biological activity in a process commonly referred to herein as photobiomodulation (“PBM”). PBM involves the use of visible light to near infrared light (NIR) (500-1000 nm) produced by a laser or a non-coherent light source applied to the surface of the body to produce beneficial effects in a wide range of disease states. Chung et al., Ann. Biomed. Eng (2011); Hashmi et al., PM. R. 2: S292-S305 (2010); Rojas et al., Dovepress 2011:49-67 (2011); and Tata and Waynant, Laser and Photonics Reviews 5:1-12 (2010). PBM requires the use of light with a suitable intensity, energy, and wavelengths, without significantly causing damage to the cells.

The mechanism of PBM at the cellular level has been ascribed to the activation of mitochondrial respiratory chain components resulting in stabilization of metabolic function. A growing body of evidence suggests that cytochrome C oxidase (CCO) is a key photoacceptor of light in the far red to near infrared spectral range. Grossman et al., Lasers. Surg. Med. 22:212-218 (1998); Karu et al., J. Photochem. Photobiol. B. 27:219-223 (1995); Karu and Kolyakov, Photomed. Laser Surg. 23:355-361 (2005); Karu et al., Lasers Surg. Med. 36:307-314 (2005); and Wong-Riley et al., J. Biol. Chem. 280:4761-4771 (2005).

There are many disorders including trauma or diseases that can afflict the eye. Ocular disease can include, for example, glaucoma, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, central serous retinopathy (CRS), non-arteritis anterior ischemic optic neuropathy (NAION), Leber's hereditary optic neuropathy disease, uveitis, and the like. Other disorders can include physical trauma (e.g., cataract or lens surgery) or other sources of ocular damage or degeneration. Ocular degeneration can include the process of cell destruction resulting from a primary destructive event such as ocular trauma or surgery, as well as from secondary, delayed and progressive destructive mechanisms that are invoked by cells due to the occurrence of a primary destructive or disease event.

It is desirable to develop methods and devices for treatment of these ocular diseases, disorders, or degeneration. In particular, it is desirable to develop methods and devices for treatment that may be less invasive or have fewer side effects than surgery or pharmacological treatments or which can be used in conjunction with surgery or pharmacological treatments to aid in healing or treatment.

SUMMARY OF THE DISCLOSURE

The multi-wavelength phototherapy systems and methods, which are described in further detail herein, are based upon the discovery that certain cellular responses, including cellular responses within a damaged and/or diseased tissue, can be promoted through the coordinated and targeted delivery to a cell of light having two distinct wavelengths, wherein a first dose of light having a first wavelength (or range of wavelengths) can stimulate a first intracellular activity and a second dose of light having a second wavelength (or range of wavelengths) can stimulate a second intracellular activity. Moreover, certain therapeutic benefits can be achieved for a patient afflicted with a damaged and/or diseased tissue by promoting a desired cellular response that contributes to the healing of a damaged tissue and/or reversal or slowing of disease progression in a diseased tissue.

Within certain embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods for improving and/or restoring one or more functionality of a target cell, which systems and methods include the coordinated and targeted delivery to a cell and/or tissue of two or more distinct wavelengths of light to stimulate the activity of two or more light sensitive factors or photoacceptors thereby improving and/or restoring target cell functionality, in particular a functionality of a target cell that is associated with a disorder and/or disease.

Within other embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods for stimulating cytochrome c oxidase (CCO) activity in a cell and/or tissue, which methods include the coordinated and targeted delivery of two or more doses of light to a cell having two or more light sensitive factors that are associated with, and necessary for, CCO activity, wherein a first light dose has a first wavelength that can activate a first light sensitive factor in CCO and a second light dose has a second wavelength that can activate a second light sensitive factor in CCO thereby stimulating CCO activity. Within certain aspects of these embodiments, stimulating CCO activity improves and/or restores the functionality of a target cell, in particular a target cell within a target tissue, such as a target tissue having one or more cell that is associated with a disorder and/or disease, certain aspects of which can be reversed and/or the progression of which can be slowed by increasing intracellular CCO activity.

Within further embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods for the treatment of a patient afflicted with a disorder and/or a disease that is associated with one or more absent or diminished cellular functionality, wherein the systems and methods include the coordinated and targeted delivery of two or more distinct wavelengths of light to one or more cells in the patient to restore the absent cellular functionality and/or enhance the diminished cellular functionality thereby treating the disorder and/or disease. Within certain aspects of these embodiments, the absent or diminished cellular functionality includes an intracellular functionality, such as intracellular CCO activity, which in a target cell having two or more light sensitive factors that are associated with, and necessary for, the intracellular functionality.

Within yet other embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods for the treatment of a patient afflicted with an ocular disorder and/or a disease that is associated with one or more absent and/or diminished functionality in an ocular cell, the systems and methods including the coordinated and targeted delivery of two or more distinct wavelengths of light to an eye in the patient to restore and or enhance the absent and/or diminished functionality to the ocular cell thereby treating the ocular disorder and/or disease.

Within certain aspects of these systems and methods the ocular disorder and/or disease is an acute or chronic ocular disorder and/or disease, which includes, for example, an ocular degenerating disease, such as blurred or loss of vision, visual acuity impairment, inflammation, and deterioration in contrast sensitivity.

Within other aspects of these systems and methods the ocular disorder and/or disease is an ocular syndrome such as, for example, glaucoma, age-related macular degeneration (AMD) including either dry or wet, diabetic retinopathy, retinitis pigmentosa, CRS, NAION, Leber's disease, ocular surgery, uveitis, hypertensive retinopathy, or a process that interferes with one or more function of an eye via a vascular or neurological mechanism, and optic neuritis.

Within further aspects of these systems and methods the ocular disorder and/or disease is an acute or chronic ocular eyelid disease including bleparitis, periorbital wrinkles, seborrhea, or other eyelid skin condition such as, for example, psoriasis and eczema.

Within yet other aspects of these systems and methods the ocular disorder and/or disease is an acute or chronic ocular conjunctiva or corneal disease including an acute injury such as exposure keratitis or UV keratitis, dry eyes, viral infections, bacterial infections, corneal abrasions, corneal edema, surgical incisions, perforating injuries, episcleritis or scleritis.

Within still further aspects of these systems and methods the ocular disorder and/or disease is an acute or chronic anterior chamber and vitreous disease including iritis, vitritis, endophthalmitis (bacterial and sterile).

These and other aspects of the present disclosure will be best understood in conjunction with the following drawings, which exemplify certain aspects of the various embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of Contrast Sensitivity at 1.5 cycles per degree (cpd; in log units) prior to treatment (0) and at 2, 4, 6, and 12 months showing an average for all patients during a course of multi-wavelength photobiomodulation therapy according to the systems and methods of the present disclosure (described in Example 1). N=18. Repeated measures ANOVA for Contrast Sensitivity (1.5 cycles/degree): F (4,68)=4.39, p less than 0.0032. (i.e., statistically significant).

FIG. 2 is a bar graph of Contrast Sensitivity at 3 cpd (in log units), which shows an average contrast sensitivity score for all patients prior to treatment (0) and at 2, 4, 6, and 12 months post-treatment with a course of multi-wavelength photobiomodulation therapy according to the systems and methods of the present disclosure (described in Example 1). N=18. Repeated measures ANOVA for Contrast sensitivity (3 cycles/degree): F (4,68)=11.44, p less than 0.0001. (i.e., statistically significant).

FIG. 3 is a bar graph of ETDRS Visual Acuity (in log MAR units), which shows an average Log MAR ETDRS score for all patients prior to treatment (0) and at 2, 4, 6, and 12 months post-treatment with a course of multi-wavelength photobiomodulation therapy according to the systems and methods of the present disclosure (described in Example 1). N=18. Repeated Measures ANOVA yielded F (4.68)=18.86, p less than 0.0001. (i.e., statistically significant).

FIGS. 4A and 4B are ocular coherence tomography (OCT) data (representative of the data described in Example 2 and summarized in Table 1) that shows retinal scan and sections (FIG. 4A) and retinal thickness (FIG. 4B), in particular at the central macula, in a patient afflicted with dry adult-onset macular degeneration (dry AMD) who underwent a course of multi-wavelength photobiomodulation therapy according to the systems and methods of the present disclosure.

FIG. 5 is a graph showing the percentage of patient's eyes achieving visual acuity (VA) ETDRS Line improvement following a 3× per week for 3-week treatment. T-test comparison between the pretreatment baseline VA mean letter score versus the VA mean letter score following 3-week treatment was statistically significant, p<0.05. N=41 eyes.

FIG. 6. is a graph showing a change in visual acuity (VA) letter score (change from background) for individual patients following a 3× per week for 3-week treatment. T-test comparison between the pretreatment baseline VA letter score versus the VA letter score following 3-week treatment was statistically significant, p<0.05. N=41 eyes.

FIG. 7. is a graph showing a reduction in anatomical pathology (drusen volume) following a photobiomodulation (“PBM”) therapy protocol according the methods disclosed herein. Data are from individual patients following a 3× per week for 3-week treatment. T-test comparison between the pretreatment baseline Drusen volume versus the Drusen volume following 3-week treatment was statistically significant, p<0.05. N=41 eyes. These data demonstrate a therapeutic benefit of PBM therapy according to the methods of the present disclosure.

FIG. 8 is a schematic flowchart of one embodiment of a multi-wavelength phototherapy system and method for improving or restoring a functionality of a target cell, through the coordinated and targeted delivery to a cell of two or more distinct wavelengths of light to stimulate the activity of two or more light sensitive factors thereby improving or restoring target cell functionality.

FIG. 9 is a schematic flowchart of one embodiment of a multi-wavelength phototherapy system and method for stimulating cytochrome c oxidase (CCO) activity in a cell through the coordinated and targeted delivery of two or more doses of light to a cell having two or more light sensitive factors that are associated with, and necessary for, CCO activity, wherein a first light dose has a first wavelength that can activate a first light sensitive factor in CCO and a second light dose has a second wavelength that can activate a second light sensitive factor in CCO thereby stimulating CCO activity.

FIG. 10 is a schematic flowchart of one embodiment of a multi-wavelength phototherapy system and method for the treatment of a patient afflicted with a disorder or disease that is associated with one or more absent or diminished cellular functionality through the coordinated and targeted delivery of two or more distinct wavelengths of light to one or more cells in the patient to restore the absent cellular functionality or enhance the diminished cellular functionality thereby treating the disorder or disease.

FIG. 11 is a schematic flowchart of one embodiment of a multi-wavelength phototherapy system and method for the treatment of a patient afflicted with an ocular disorder or disease that is associated with one or more absent or diminished functionality in an ocular cell, the systems and methods including the coordinated and targeted delivery of two or more distinct wavelengths of light to an eye in the patient to restore and or enhance the absent or diminished functionality to the ocular cell thereby treating the ocular disorder or disease.

FIG. 12 is a drawing showing the principle measurement locations used in the cadaver study disclosed herein and described in detail in Example 4.

FIG. 13 is a graph showing mean fluence rates (open eye) that were obtained from the cadaver study disclosed herein and described in detail in Example 4.

FIG. 14 is a graph showing mean fluence rates (closed eye) that were obtained from the cadaver study disclosed herein and described in detail in Example 4.

DETAILED DESCRIPTION

The present disclosure is based upon the discovery that therapeutic benefits can be achieved for a patient afflicted with damaged and/or diseased tissue by promoting one or more cellular responses within a cell of a damaged and/or diseased tissue, which cellular responses can be promoted through the coordinated and targeted delivery of two or more doses of light, wherein a first dose of light has a first wavelength or range of wavelengths, which can stimulate a first intracellular activity, and a second dose of light has a second wavelength or range of wavelengths, which can stimulate a second intracellular activity, wherein the coordinated stimulation of the first and second intracellular activities promotes a desired cellular response thereby facilitating healing of the damaged tissue and/or reversing or slowing disease progression in the diseased tissue.

In the non- or minimally-invasive multi-wavelength phototherapy systems and methods disclosed herein, an efficacious amount of light energy is delivered to an internal tissue, as exemplified herein by an ocular tissue, from one or more light sources that are: (1) exterior to the body (i.e., non-invasive) or that is in a subcutaneous location within the body (i.e., minimally-invasive) and (2) capable of producing light having a distinct and specified wavelength and/or range of wavelengths. In particular, the multi-wavelength phototherapy systems and methods disclosed herein employ light having a first wavelength or range of wavelengths that can stimulate a first light sensitive factor and light having a second wavelength or range of wavelengths that can stimulate a second light sensitive factor, wherein the targeted and coordinated delivery of light having a first wavelength and light having a second wavelength promotes a cellular response within a targeted tissue thereby facilitating the healing of the damaged tissue and/or reversing or slowing the progression of disease in the diseased tissue.

As described in further detail herein, such multi-wavelength phototherapy systems and methods provide a therapeutic benefit when delivered alone, which therapeutic benefit can be further enhanced when those systems and methods are used in combination with one or more small molecule drug and/or biologic and/or when used in combination with a second therapeutically suitable device and/or other treatment regimen, which drugs, biologics, devices, and/or treatment regimen can be administered to a patient prior to, concurrently with, and/or subsequent to the targeted and coordinated delivery of the multi-wavelength phototherapy.

Light exhibiting a single wavelength or range of wavelengths such as, for example, red light having a wavelength of 600-700 nm or near-infrared light (“NIR”) having a wavelength of 800-900 nm can be used to stimulate mitochondrial cytochrome c oxidase (“CCO”) enzymatic activity. As disclosed herein, the targeted and coordinated use of two or more sources of light, each light source having a distinct wavelength and intensity, can yield a unique, additive, or synergistic therapeutic benefit that is substantially improved. Within some aspects, the therapeutic benefit may be greater than simply the therapeutic benefit exhibited by each wavelength of light when delivered in isolation and/or when delivered in a non-targeted, non-coordinated fashion to the cell and/or tissue of interest.

More specifically, distinct wavelengths of light can, for example, stimulate structurally and functionally distinct moieties within a protein of a target cell, such as the CuA and CuB moieties of a mitochondrial cytochrome c oxidase (“CCO”). It is recognized as part of the present disclosure that by coordinating temporally the delivery of two or more wavelengths of light to the CuA and CuB moieties of a mitochondrial CCO, the electron flow and oxygen binding via the CCO enzyme can be independently, sequentially, or in combination optimized to: (a) substantially improve overall CCO activity, (b) restore mitochondrial membrane potential (“MMP”), and (c) increase the level of ATP synthesis. Moreover, such temporally coordinated and targeted delivery of multiple wavelengths of light substantially improves the therapeutic efficacy of previously-described, single wavelength phototherapy systems and methods.

The multi-wavelength phototherapy systems and methods of the present disclosure can, therefore, be used advantageously to restore mitochondrial membrane potential (MMP) and/or to increase ATP formation in a damaged and/or diseased tissue, which damaged and/or diseased tissue exhibits a characteristic reduction in its access to oxygen.

As described in further detail herein, the present disclosure contemplates, for example, the targeted and coordinated delivery of light having a wavelength of from about 640 nm to about 700 nm to activate a CCO CuB moiety thereby displacing one or more CCO inhibitors (such as, e.g., the vasodilator NO) that occupy one or more CCO oxygen binding sites. The localized release of NO from mitochondria can, therefore, be exploited to improve local blood flow thereby increasing O₂ and nutrient levels in a damaged and/or a diseased tissue. The targeted delivery of light having a wavelength of from about 640 nm to about 700 nm may also be employed to preferentially increase the O₂ binding affinity at a CCO active site thereby stimulating electron transport and aerobic generation of ATP.

As a further example, the present disclosure also provides the delivery of near infrared (“NIR”) light (i.e., light having a wavelength of from about 800 nm to about 900 nm), which NIR light exhibits therapeutic benefit by facilitating the photo-mediated transfer of electrons from cytochrome C to CCO thereby improving the efficiency of electron flow and restoring mitochondrial membrane potential (MMP).

Thus, the present disclosure provides systems and methods that employ light having two or more distinct wavelengths, or ranges of wavelengths, which systems and methods include the coordinated delivery, which includes both concurrent delivery and temporally coordinated delivery, of multiple wavelengths of light with predefined optical parameters, such as, e.g., duration of delivery, frequency of delivery, continuous delivery, pulsed delivery, and fluence level of delivery, to provide, thereby, an individualized and personalized treatment regimen, which is optimized to restore, promote, and/or enhance mitochondrial function and, as a consequence, facilitate the recovery of a damaged tissue and/or reverse or slow the progression of disease in a diseased tissue.

As described herein, various aspects of such multi-wavelength phototherapy systems and methods may be tailored to affect important intracellular mediators such as, e.g., ATP, GTP, Nitric Oxide (NO), and/or reactive oxidative species (ROS), each of which is used by a cell to transmit intracellular stimuli via one or more signal transduction pathways, which, in turn, regulate downstream cellular activities/functionalities.

The capacity of the systems and methods of the present disclosure to control such second messenger-mediated cellular pathways provides an opportunity to affect key regulatory mechanisms of cell activity. Protein kinases, for example, represent a major class of enzymes that lead to the phosphorylation of protein targets. ATP, which is the active substrate for protein kinases, transfers a high-energy phosphorous bond to target proteins. Protein activity can, therefore, be increased or decreased by the phosphorylation of a target protein at one or more sites. As a consequence, enzyme activities and/or cellular pathways can be controlled by the availability of ATP to and the level of ATP within a cell, such as can be achieved by the inhibition or activation of one or more protein targets by one or more protein kinases.

As part of the present disclosure, it was discovered that the use of multiple wavelengths of light provides unique opportunities for treating damaged and/or diseased tissue by regulating signal transduction, mediating protein kinase activity, improving cellular performance, and restoring cellular function.

The multi-wavelength phototherapy systems and methods disclosed herein can be readily adapted for regulating and controlling cellular gene expression and restoring cellular function in a damaged and/or diseased tissue. Gene expression patterns are used by cells to coordinate and regulate numerous pathways that influence subsequent cellular activity. Multi-wavelength phototherapy systems and methods can, for example, be adapted for use in changing a gene expression pattern for multiple genes involved in cellular metabolism. Up regulation of several genes involved in electron chain transport, energy metabolism, and oxidative phosphorylation can be exploited to rejuvenate a cell's metabolic capacity and/or to stimulate ATP production, which can drive other pleiotropic processes and, collectively, can facilitate long-term improvement in and/or normalization of one or more cellular functions. In a related aspect, the multi-wavelength phototherapy systems and methods disclosed herein can also be adapted to affect NFkβ, a major cellular regulator of inflammatory pathways and gene expression.

Based upon these and other discoveries, which are described in detail herein, the present disclosure provides:

-   -   1. Multi-wavelength phototherapy systems and methods for         stimulating cytochrome c oxidase (CCO) activity in a cell and/or         tissue, which methods include the coordinated delivery of two or         more doses of light to a cell having two or more light sensitive         factors that are associated with CCO activity, wherein each         light dose has a distinct wavelength or range of wavelengths,         wherein a first wavelength of light can stimulate a first light         sensitive factor and a second wavelength of light can stimulate         a second light sensitive factor thereby improving and/or         restoring the one or more functionality of the target cell, in         particular a target cell within a target tissue;     -   2. Multi-wavelength phototherapy systems and methods for         improving and/or restoring one or more functionality of a target         cell, which systems and methods include the coordinated and         targeted delivery to a cell and/or tissue of two or more         distinct wavelengths of light to stimulate the activity of two         or more light sensitive molecules thereby improving and/or         restoring the one or more functionality of the target cell, in         particular a target cell within a target tissue;     -   3. Multi-wavelength phototherapy systems and methods for the         treatment of a patient afflicted with a disorder and/or a         disease that is associated with one or more absent and/or         diminished cellular functionality, the systems and methods         including the coordinated and targeted delivery of two or more         distinct wavelengths of light to one or more cells in the         patient thereby restoring the absent cellular functionality         and/or enhancing the diminished cellular functionality thereby         treating the disorder and/or disease; and     -   4. Multi-wavelength phototherapy systems and methods for the         treatment of a patient afflicted with an ocular disorder and/or         a disease that is associated with one or more absent and/or         diminished functionality in an ocular cell, the systems and         methods including the coordinated and targeted delivery of two         or more distinct wavelengths of light to an eye in the patient         to restore and or enhance the absent and/or diminished         functionality to the ocular cell thereby treating the ocular         disorder and/or disease.

These and other aspects of the present disclosure can be better understood by reference to the following non-limiting definitions.

DEFINITIONS

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter. As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

It will be understood that, unless indicated to the contrary, terms intended to be “open” (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Phrases such as “at least one,” and “one or more,” and terms such as “a” or “an” include both the singular and the plural. Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

Conditional language, for example, among others, “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment.

It will be further understood that where features or aspects of the disclosure are described in terms of Markush groups, the disclosure is also intended to be described in terms of any individual member or subgroup of members of the Markush group.

All references cited herein, whether supra or infra, including, but not limited to, patents, patent applications, and patent publications, whether U.S., PCT, or non-U.S. foreign, and all technical and/or scientific publications are hereby incorporated by reference in their entirety.

As used herein, the term “phototherapy,” refers to the therapeutic delivery of light energy to a subject (e.g., a human or other mammal) to achieve one or more therapeutic benefits. The term “phototherapy,” encompasses the term “Low level light therapy” or “LLLT,” which refers to the therapeutic delivery of light energy at an irradiance level that is at or above an irradiance level that can promote one or more desired biostimulatory effects and is below an irradiance level that can cut, cauterize, and/or ablate a biological tissue. See, e.g., U.S. Pat. Nos. 6,537,304 and 6,918,922, each of which is incorporated by reference herein in its entirety.

As used herein, the term “light source” refers to an element of the light therapy apparatus (a/k/a phototherapy apparatus) that is configured to provide an optical output (e.g., to transmit light from a light therapy apparatus to a target tissue, such as an ocular tissue, of a patient). As used herein, the term “red light” refers to light having a wavelength of from about 640 nm to about 700 nm and the term “near infrared light” or “NIR” refers to light having a wavelength of from about 800 nm to about 900 nm.

As used herein, the terms “cytochrome c oxidase,” “CCO,” “Complex IV,” and “EC 1.9.3.1” refer to a large transmembrane protein complex that is produced in the mitochondria and located within the mitochondrial membrane of eukaryotic cells. CCO is the last enzyme in the mitochondrial respiratory electron transport chain of mitochondria—receiving an electron from each of four cytochrome c molecules, transferring them to an oxygen molecule, and converting molecular oxygen to two molecules of water. CCO binds four protons from the inner aqueous phase to make water and translocates four protons across the membrane, thereby establishing a transmembrane proton electrochemical potential, which is used by ATP synthase for the synthesis of ATP.

As used herein, the terms “treatment,” “treating,” “therapeutic regimen,” and “treatment regimen” refer, generally, to a therapeutic systems and methods that promote the healing of a damaged tissue and/or reverse or slow the progression of disease in a diseased tissue, which can be achieved by restoring one or more functionality in a cell within the damaged and/or diseased tissue. The terms “treatment,” “treating,” “therapeutic regimen,” and “treatment regimen” include protocols and associated procedures that are used to provide a therapeutic system or method that includes one or more periods during which light is irradiated to one or more targeted cells and tissues, including ocular cells and tissues.

As used herein, the terms “target,” “target area,” and “target region” refer to a particular ocular area, region, location, structure, population, or projection within a tissue, such as a retina or an optic nerve, to which light is delivered in association with the treatment of a particular condition, disease, disorder, or injury, such as a condition, disease, disorder, or injury of an eye. In certain embodiments, the irradiated portion of a tissue, such as an eye can include the entire tissue. In other embodiments, the irradiated portion of the tissue can include a targeted region of the tissue, such as the retinal region, the macula, or the cornea of an eye.

As used herein, the term “degeneration” refers, generally, to a process of cell destruction resulting from primary destructive events such as trauma or surgery, as well as from secondary, delayed and progressive destructive mechanisms that are invoked by cells due to the occurrence of the primary destructive or disease event.

As used herein, the term “primary destructive events” refers to disease processes or physical injury or insult, including surgery, but also include other diseases and conditions, which, in the case of ocular disorders and diseases, can include glaucoma, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, CRS, NAION, Leber's disease, ocular surgery, uveitis, cerebral ischemia including focal optic nerve ischemia, and physical trauma such as crush or compression injury to ocular tissues, including a crush or compression injury of the optic nerves or retina, or any acute injury or insult producing ocular degeneration.

As used herein, the term “secondary destructive mechanisms” refers to any mechanism that leads to the generation and release of neurotoxic molecules, including but not limited to, apoptosis, depletion of cellular energy stores because of changes in mitochondrial membrane permeability, release or failure in the reuptake of excessive glutamate, free radical damage, reperfusion injury, deposition of insoluble proteins including lipofuscin and β-amyloid and activity of complement, cytokines, and inflammatory conditions.

As used herein, the term “cytoprotection” refers to a therapeutic strategy for slowing or preventing the otherwise irreversible loss of a target tissue, such as an ocular tissue, due to degeneration after a primary destructive event, whether the tissue degeneration loss is due to disease mechanisms associated with the primary destructive event or secondary destructive mechanisms.

Both primary and secondary mechanisms contribute to forming a “zone of danger” wherein a tissue within the zone of danger that has survived a primary destructive event remains at a risk of dying due to one or more processes having a delayed effect.

As used herein, the term “restoration” refers to an increase in a functionality of a cell, such as a cell from a damaged and/or diseased tissue, to a level that is comparable to, equal to, or higher than the functionality of a comparable normal cell, such as a cell from a comparable undamaged and disease-free tissue, such as a tissue from a healthy individual.

As used herein, “significance” or “significant” relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is “significant” or has “significance”, statistical manipulations of the data can be performed to calculate a probability, expressed as a “p-value”. Those p-values that fall below a user-defined cutoff point are regarded as significant. A p-value in some embodiments less than or equal to 0.05, in some embodiments less than 0.01, in some embodiments less than 0.005, and in some embodiments less than 0.001, are regarded as significant.

As used herein, the term “diagnosed” refers to a determination that has been made regarding a damaged and/or diseases tissue. A diagnosis may be made prior to using or performing the present multi-wavelength phototherapy systems and methods.

Multi-Wavelength Phototherapy Systems and Methods

As described in further detail herein, it was discovered as part of the present disclosure that the coordinated and targeted delivery to a cell or tissue of light having two or more specified and distinct wavelengths (or ranges of wavelengths) can be advantageously employed: (a) to improve intracellular mitochondrial function via increased cytochrome C oxidase (“CCO”) activity, (b) to restore an intracellular mitochondrial membrane potential (“MMP”), and (c) to up-regulate intracellular ATP synthesis. Moreover, such enhanced intracellular activities may be further exploited to promote a localized cellular response including, e.g., a cellular response that is absent from or present at an insufficient level in a damaged and/or diseased tissue as compared to a corresponding normal, undamaged, and/or healthy tissue.

Thus, within certain embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods for promoting a desired cellular response, which methods include the coordinated and targeted delivery to a cell of two or more doses of light, wherein a first dose of light has a first wavelength or range of wavelengths that can stimulate a first intracellular activity and a second dose of light has a second wavelength or range of wavelengths that can stimulate a second intracellular activity, wherein the coordinated and targeted delivery of the first and second doses of light promotes the desired cellular response.

Within related embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods for the treatment of damaged and/or diseased tissue, which methods include the coordinated and targeted delivery to a damaged and/or diseased tissue of two or more doses of light wherein a first dose of light has a first wavelength or range of wavelengths that can stimulate a first intracellular activity and a second dose of light has a second wavelength or range of wavelengths that can stimulate a second intracellular activity, wherein the coordinated and targeted delivery of the first and second doses of light can promote a desired cellular response within the damaged and/or diseased tissue thereby promoting the healing of the damaged tissue and/or reversing or slowing the progression of disease in the diseased tissue.

Within certain aspects of these embodiments, the present disclosure is exemplified by multi-wavelength phototherapy systems and methods for the treatment of damaged and/or diseased ocular tissue, which methods include the coordinated and targeted delivery to damaged and/or diseased ocular tissue within an eye of two or more doses of light, wherein a first dose of light has a first wavelength or range of wavelengths that can stimulate a first intracellular activity within the damaged and/or diseased ocular tissue, wherein a second dose of light has a second wavelength or range of wavelengths that can stimulate a second intracellular activity within the damaged and/or diseased ocular tissue, and wherein the coordinated and targeted delivery of the first and second doses of light can promote a desired cellular response within the damaged and/or diseased ocular tissue thereby promoting the healing of the damaged ocular tissue and/or reversing or slowing the progression of disease in the diseased ocular tissue.

FIG. 8 illustrates one embodiment of a multi-wavelength phototherapy system and method for improving or restoring a functionality of a target cell or tissue through the coordinated and targeted delivery to a cell or tissue of two or more distinct wavelengths of light to stimulate the activity of two or more light sensitive factors thereby improving or restoring target cell functionality. By these systems and methods, a first light source is positioned for targeted delivery of a first wavelength of light to a target cell 602; a second light source is positioned for targeted delivery of a second wavelength of light to the target cell 604; a first light sensitive factor in the target cell is activated with the first wavelength of light 606; and a second light sensitive factor is activated in the target cell with the second wavelength of light 608.

FIG. 9 illustrates one embodiment of a multi-wavelength phototherapy system and method for stimulating cytochrome c oxidase (CCO) activity in a cell through the coordinated and targeted delivery of two or more doses of light to a cell having two or more light sensitive factors that are associated with, and necessary for, CCO activity, wherein a first light dose has a first wavelength that can activate a first light sensitive factor in CCO and a second light dose has a second wavelength that can activate a second light sensitive factor in CCO thereby stimulating CCO activity. By these systems and methods, a first light source is positioned for targeted delivery of a first wavelength of light to a target cell that is producing cytochrome C oxidase 702; a second light source is positioned for targeted delivery of a second wavelength of light to the target cell that is producing cytochrome C oxidase 704; a first cytochrome C oxidase associated light sensitive factor in the target cell is activated with the first wavelength of light 706; and a second cytochrome C oxidase associated light sensitive factor is activated in the target cell with the second wavelength of light 708.

FIG. 10 illustrates one embodiment of a multi-wavelength phototherapy system and method for the treatment of a patient afflicted with a disorder or disease that is associated with one or more absent or diminished cellular functionality through the coordinated and targeted delivery of two or more distinct wavelengths of light to one or more cells in the patient to restore the absent cellular functionality or enhance the diminished cellular functionality thereby treating the disorder or disease. By these systems and methods, a first light source is positioned for targeted delivery of a first wavelength of light to a target cell that is associated with a disorder or disease in a patient afflicted with the disorder or disease 802; a second light source is positioned for targeted delivery of a second wavelength of light to the target cell that is associated with a disorder or disease in a patient afflicted with the disorder or disease 804; a first light sensitive factor in the disorder or disease associated target cell is activated with the first wavelength of light 806; and a second light sensitive factor in the disorder or disease associated cell is activated with the second wavelength of light 808.

FIG. 11 illustrates one embodiment of a multi-wavelength phototherapy system and method for the treatment of a patient afflicted with an ocular disorder or disease that is associated with one or more absent or diminished functionality in an ocular cell, the systems and methods including the coordinated and targeted delivery of two or more distinct wavelengths of light to an eye in the patient to restore and or enhance the absent or diminished functionality to the ocular cell thereby treating the ocular disorder or disease. By these systems and methods, a first light source is positioned for targeted delivery of a first wavelength of light to a target cell that is associated with an ocular disorder or disease in a patient afflicted with the ocular disorder or disease 902; a second light source is positioned for targeted delivery of a second wavelength of light to the target cell that is associated with the ocular disorder or disease in a patient afflicted with the ocular disorder or disease 904; a first light sensitive factor in the ocular disorder or disease associated target cell is activated with the first wavelength of light 906; and a second light sensitive factor in the ocular disorder or disease associated cell is activated with the second wavelength of light 908.

Multi-wavelength phototherapy systems and methods for the treatment of damaged and/or diseased ocular tissue are exemplified herein by multi-wavelength phototherapy systems and methods for the treatment of an ocular disorder and/or an ocular disease, treatments restore and/or enhance one or more symptom of an ocular disorder and/or disease including glaucoma, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, CRS, NAION, Leber's disease, ocular damage resulting from a surgical procedure, and/or uveitis.

In certain aspects of these embodiments, light may be delivered through a closed eyelid, in which much of the light can be expected to scatter over a relatively broad area of the retina, or it may be administered to the open eye. In the case of the open eye, the majority of the therapeutic light can be delivered to the retina through the lens and pupil of the eye with minimal scattering. This can be accomplished, for example, through a Spatial Light Modulator (SLM) that precisely shapes and controls the exposed area on the retina. The SLM may be an LCOS panel, scanning mirror, deformable mirror array, or other modulation device.

1. Light Parameters for Achieving Therapeutic Efficacy

The various parameters of a light beam that is emitted from a light source can be advantageously selected to provide therapeutic benefit while controlling, inhibiting, preventing, minimizing, or reducing injury and/or discomfort to a patient, which can result from the light-induced heating of a target tissue, such as skin or eye tissue. These various parameters can be selected by those having skill in the art and can be combined within the range of values that are disclosed herein to achieve suitable conditions for the treatment of tissue damage or disease in accordance with the present systems and methods.

These light beam parameters can include, but are not limited to: (1) the wavelength of each of the two or more sources of light, (2) the irradiance or power density of each of the two or more sources of light, (3) the temporal pulse width and shape, duty cycle, repetition rate, and irradiance per pulse for each of the two or more sources of light, and (4) the total treatment time with each of the two or more sources of light. Following is a description of each of these parameters and guidance on the selection of those parameters for use in the multi-wavelength systems and methods disclosed herein.

a. Wavelengths or Ranges of Wavelengths

In certain embodiments, light in the visible to near-infrared wavelength range can be delivered to a target cell or tissue, such as a patient's skin or eye tissue. The light can be substantially monochromatic (i.e., having a single wavelength or a narrow band of wavelengths) wherein the desired cellular response can be established with the use of two or more selected wavelengths of light.

For example, one source of light can have a wavelength of from about 550 nanometers to about 1064 nanometers or from about 590 nanometers to about 980 nanometers. A plurality of wavelengths of light can be employed wherein a first wavelength of light is delivered concurrently with a second wavelength of light or wherein a first wavelength of light is delivered independently from and sequentially to a second wavelength of light.

In certain aspects of the present phototherapy systems and methods, the light can have a wavelength distribution that exhibits a peak wavelength wherein the wavelength distribution has a line width of less than ±10 nanometers from the peak wavelength, or less than ±4 nanometers from the peak wavelength, full width at 90% of energy. In related aspects, each wavelength of light can be selected independently from 590 nm±10%, 670 nm±10%, 810 nm±10%, and 1064 nm±10%, with a spectral line width of less than 4 nanometers, full width at 90% of energy. In further aspects, each wavelength of light can be selected independently from a wavelength distribution peaked at a peak wavelength and having a line width of less than ±40 nanometers from the peak wavelength at 50% of energy. In yet other aspects, each wavelength of light can be selected independently from 590 nm±10%, 670 nm±10%, 810 nm±10%, and 1064 nm±10%, with a spectral line width of less than 40 nanometers, full width at 50% of energy.

To ensure that the amount of light transmitted to the treated cell or tissue is maximized, each preselected wavelength of light can be selected to be at or near a transmission peak (or at or near an absorption minimum) for the intervening tissue. For example, a first wavelength can correspond to a peak in the transmission spectrum of tissue at about 820 nanometers (NIR) and a second wavelength can correspond to a peak in the transmission spectrum of tissue at about 670 nanometers (red visible).

The present phototherapy systems and methods can be performed with a light source having one or more continuously-emitting GaAlAs laser diodes each having a wavelength as described herein. Alternatively, the present methods can be performed with a light source having one or more LED(s), each of which providing non-coherent light having a wavelength as described herein.

Each of the two or more wavelengths of light can be selected to stimulate or activate one or more photoacceptors within a target cell or tissue. Without being bound by theory or specific mechanism of action, it is believed that delivery of light to one or more CCO photoacceptors, for example, will increase the production of ATP in the target cell or tissue to, thereby, control, inhibit, prevent, minimize, or reduce apoptosis of a damaged tissue, thus producing a beneficial therapeutic effect as described in detail herein. Wavelengths may also be chosen to activate one or more photoacceptors to control, inhibit, or stimulate distinct biological responses in a target cell or tissue.

Some photoacceptors, such as water or hemoglobin, are ubiquitous and absorb light to such a degree that light energy cannot reach a target tissue. It is known, for example, that water absorbs light at wavelengths above approximately 1300 nanometers. Thus, light at those wavelengths cannot penetrate effectively a target tissue due to the water content. Water is, however, transparent or nearly transparent to light at wavelengths of from about 300 nanometers to about 1300 nanometers. Similarly, hemoglobin, which absorbs light from about 300 nanometers to about 670 nanometers, is reasonably transparent to light above about 670 nanometers. Based upon these known factors that restrict the effective delivery of light, an “IR window” can be defined for the penetration of light that is delivered to a target tissue. Within this IR window, certain wavelengths of light can penetrate with less restriction by light absorbing molecules such as, for example, water and hemoglobin.

b. Irradiances and Power Densities

Within certain aspects of the present phototherapy systems and methods, light sources may be employed that emit a light beam having a time-averaged irradiance, or power density, at the emission surface of the light sources (e.g., at the tissue surface, such as a retinal surface) of from about 0.005 mW/cm² to about 10 W/cm², or from about 0.01 mW/cm² to about 5 W/cm², of from about 0.01 mW/cm² to about 1 W/cm², or from about 1 mW/cm² to about 500 mW/cm², or from about 500 mW/cm² to about 1 W/cm² across the cross-sectional area of the light beam.

Within other aspects of the present phototherapy systems and methods, light sources may be employed that emit a light beam having a time-averaged irradiance, or power density, that can be reduced generally by a factor of 1/e from the values that would be used if the light sources were applied to a closed eyelid versus directly to the retina. For example, the time-averaged irradiance at the target tissue (e.g., at a depth of approximately two centimeters below the eyelid) can be from about 0.001 mW/cm² to about 1 W/cm² at the level of the tissue or at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mW/cm², or greater at the level of the tissue depending upon the desired therapeutic application.

For a pulsed light beam, the time-averaged irradiance can be averaged over a long time period as compared to a temporal pulse width of the pulses (e.g., averaged over a fraction of a second longer than the temporal pulse width, over 1 second, or over multiple seconds). For a continuous-wave (CW) light beam with time-varying irradiance, the time-averaged irradiance can be an average of the instantaneous irradiance averaged over a time period longer than a characteristic time period of fluctuations of the light beam.

In certain aspects of the present phototherapy systems and methods, a duty cycle can be from about 1% to about 80%, or from about 10% to about 30%, or about 20% with a peak irradiance at the target tissue of from about 0.001 mW/cm² to about 1 W/cm², about 0.01 mW/cm² to about 500 mW/cm², about 10 mW/cm² to about 100 mW/cm², or about 25 mW/cm² to about 125 mW/cm². For example, a pulsed dosimetry having a 20% duty cycle, a peak irradiance at the target tissue of about 50 mW/cm² can be used. In certain embodiments, the pulsed light beam has an energy or fluence per pulse (e.g., peak irradiance multiplied by the temporal pulse width) at the emission surface of the light source of from about 0.001 μJ/cm² to about 150 J/cm², or from about 0.01 μJ/cm² to about 5 J/cm², or from about 0.1 μJ/cm² to about 1 J/cm², or from about 0.01 mJ/cm² to about 100 mJ/cm², or from about 100 mJ/cm² to about 1 J/cm².

The cross-sectional area of a light beam (e.g., a multimode beam) can be determined by using an approximation of beam intensity distribution. For example, the beam intensity distribution can be approximated by a Gaussian (1/e² measurements) or “top hat” distribution and a selected perimeter of beam intensity distribution can be used to define a boundary for the area of a light beam.

The irradiance at an emission surface can be selected to provide the desired irradiances at the target tissue. The irradiance of a light beam can be variably controlled so that the emitted light energy can be adjusted to provide a selected irradiance at the target tissue. The light beam emitted from the emission surface can be continuous, with a total radiant power of from about 4 Watts to about 6 Watts. For example, the radiant power of a light beam can be 5 Watts±20% (CW).

The peak power for pulsed light can be from about 10 Watts to about 30 Watts, such as about 20 Watts. The peak power for pulsed light multiplied by the duty cycle of the pulsed light yields an average radiant power of from about 4 Watts to about 6 Watts, such as about 5 Watts.

The irradiance of a light beam can be selected to provide a predetermined irradiance at a target tissue (e.g., at a depth of the retinal pigmented epithelial layer). The selection of an appropriate irradiance of a light beam emitted from an emission surface to achieve a desired target tissue irradiance generally takes into consideration light scattering caused by non-target intervening tissues. Further information regarding the scattering of light by tissue is provided by U.S. Pat. No. 7,303,578 and Tuchin, SPIE Press 3-11 (2000), each of which is incorporated in its entirety by reference herein.

Phototherapy for the treatment of ocular conditions (e.g., glaucoma, AMD, diabetic retinopathy, retinitis pigmentosa, CRS, NAION, Leber's disease, ocular surgery, and uveitis) is based in part on the presently disclosed discovery that irradiance or power density (i.e., power per unit area or number of photons per unit area per unit time) and energy density (i.e., energy per unit area or number of photons per unit area) of light energy applied to a target tissue substantially influence the efficacy of given phototherapy regimen. These factors are particularly relevant when designing phototherapy regimen for preserving the efficacy of surviving, but endangered, cells within a “zone of danger” that is in the region surrounding a site of primary injury.

It was further discovered, and is presented herein as part of the present disclosure, that for a given wavelength of light energy, the irradiance and/or energy density of the light delivered to a target tissue—as opposed to the total power or total energy delivered to that tissue—that is determinative of the relative therapeutic efficacy of a given phototherapy regimen.

Without being bound by theory or by any specific mechanism of action, it is believed that the light energy that is delivered within a certain range of irradiances and energy densities provides a photobiomodulatory effect on an intracellular environment, such that one or more normal mitochondrial functionality is restored in a previously non- or poorly-functioning mitochondrion, such as a mitochondrion in an at-risk cell. Such a photobiomodulatory effect may include, for example, one or more interactions with one or more photoacceptors within a target tissue, which facilitates the production of ATP and/or controls, inhibits, prevents, minimizes, or reduces apoptosis in a diseased or ageing cell or increases blood flow in an ischemic tissue or modulates release of NO, ROS or other intracellular mediators to modify gene and protein expression in the at-risk cell. The role of irradiance and exposure times is discussed, for example, in Hans et al., Lasers in Surgery and Medicine 12:528-537 (1992), which is incorporated in its entirety by reference herein.

The delivery of a cytoprotective amount of light energy can also include the selection of a surface irradiance of light energy at a tissue surface, such as the surface of an eyelid or cornea, which surface irradiance corresponds to a predetermined irradiance at a target area of the tissue (e.g., the cornea or retina of an eye). As discussed in further detail herein, light propagating through a tissue can be both scattered and absorbed by that tissue. Calculations of the irradiance to be applied to a tissue surface, such as an eyelid or corneal surface, may, therefore, take into account the attenuation of light energy as it propagates through one or more intervening, non-target tissue, to ensure that a predetermined and intended irradiance is delivered to a selected area of a target tissue, such as an ocular tissue. Factors that affect the degree of attenuation of light propagating through the skin to a tissue can include, for example, the skin thickness, the patient's age and/or gender, and the location of the target area of the tissue, particularly the depth of the target area relative to the surface of the skin or, in the case of an eye, the cornea.

Factors influencing the selection of an irradiance for delivery to a target area of a given tissue include the wavelength of the light to be applied, considerations of light-induced heating of a tissue, and a patient's clinical condition and area of the damaged and/or diseased tissue. The irradiance or power density of light energy to be delivered to a tissue target area may also be influenced by, and adjusted accordingly, if the desired phototherapy regimen is delivered in combination and/or in conjunction with one or more additional therapeutic agents such as, for example, one or more neuroprotective agents, to achieve a desired biological effect. It will be understood that the selection of light parameters such as wavelength and irradiance will be influence by the specific therapeutic agents chosen.

c. Temporal Pulse Widths and Shapes, Duty Cycles, Repetition Rates, and Irradiances Per Pulse

The systems and methods of the present disclosure further contemplate various temporal profiles of pulsed light beams that may be advantageously employed to enhance the therapeutic efficacy of a given phototherapy regimen. A temporal profile includes a plurality of pulses (P₁, P₂, . . . , P_(i)), wherein each pulse exhibits a temporal pulse width during which time period an instantaneous pulse intensity or irradiance I(t) is substantially non-zero. For example, for a pulsed light beam, pulse P₁ has a temporal pulse width (a/k/a “pulse ON time”) from time t=0 to time t=T₁, pulse P₂ has a temporal pulse width from time t=T₂ to time t=T₃, and pulse P_(i) has a temporal pulse width from time t=T_(i) to time t=T_(i+1). Pulses are temporally spaced from one another by periods of time during which the intensity or irradiance of a beam is substantially zero. For example, pulse P₁ is spaced in time from pulse P₂ by a time t=T₂−T₁ (a/k/a “pulse OFF time”). Pulse ON and pulse OFF times can be substantially equal to one another or can differ from one another.

As used herein, the term “duty cycle” refers, generally, to a pulse ON time divided by the sum of a pulse ON and a pulse OFF. Thus, a pulsed light beam has a duty cycle of less than one. A duty cycle and a temporal pulse width, together, fully define a repetition rate of a given pulsed light beam.

A pulse can have a temporal pulse shape that describes the instantaneous intensity or irradiance of the pulse I(t) as a function of time. For example, the temporal pulse shape of a pulsed light beam can be irregular and need not be the same among various pulses or the temporal pulse shape of a pulsed light beam can be substantially the same among various pulses. For example, a pulse can have a square temporal pulse shape with a substantially constant instantaneous irradiance over a pulse ON time. Peak irradiances of pulses can differ from one another or can be substantially equal to one another. Various other temporal pulse shapes (e.g., triangular and trapezoidal) are also contemplated for use in the presently disclosed systems and methods.

A rise time and a fall time can be expressed relative to a specified fraction of a peak irradiance of a pulse (e.g., time to rise/fall to 50% of the peak irradiance of a pulse). As used herein, the term “peak irradiance” of a pulse P_(i) refers, generally, to the maximum value of an instantaneous irradiance I(t) during a temporal pulse width of a pulse. The instantaneous irradiance can change or can remain substantially constant during a temporal pulse width of a pulse.

As used herein, the term “pulse irradiance” I_(P) _(i) of a pulse P_(i) refers, generally, to an integral of an instantaneous irradiance I(t) of a pulse P_(i) over a temporal pulse width of a pulse:

$I_{P_{i}} = {\underset{T_{i}}{\int\limits^{T_{i + 1}}}{{{I(t)} \cdot {t}}\text{/}\left( {T_{i + 1} - T_{i}} \right)}}$

As used herein, the term “total irradiance” I_(TOTAL) refers, generally, to the sum of a pulse irradiance of pulses:

$I_{TOTAL} = {\sum\limits_{i = 0}^{N}\; I_{P_{i}}}$

As used herein, the term “time-averaged irradiance” I_(AVE) refers, generally, to the integral of an instantaneous irradiance I(t) over a period of time T that is large compared to a temporal pulse width of a pulse:

${I_{AVE} = {\underset{0}{\int\limits^{T}}{{{I(t)} \cdot {t}}\text{/}T}}},$

wherein the integral

$\underset{0}{\int\limits^{T}}{{I(t)} \cdot {t}}$

represents the energy of a pulsed light beam.

For a plurality of square pulses with different pulse irradiances, I_(P) _(i) , and different temporal pulse widths, ΔT_(i), the time-averaged irradiance over time T is defined as follows:

$I_{AVE} = {\frac{1}{T}{\sum\limits_{i}{{I_{P_{i}} \cdot \Delta}\; T_{i}}}}$

For a plurality of square pulses with equal pulse irradiances I_(P), equal temporal pulse widths, and equal pulse OFF times (having a duty cycle D), the time-averaged irradiance is defined as follows:

I _(AVE) =I _(P) ·D

Pulse irradiances and duty cycles can be selected to provide a predetermined time-averaged irradiance. In certain applications of the present systems and methods in which the time-averaged irradiance is equal to the irradiance of a continuous-wave (CW) light beam, the pulsed light beam and the CW light beam have equivalent photon and/or flux numbers. For example, a pulsed light beam having a pulse irradiance of 5 mW/cm² and a duty cycle of 20% provides the same number of photons as a CW light beam having an irradiance of 1 mW/cm². In contrast to a CW light beam, however, the parameters of a pulsed light beam can be selected such that photons are delivered in a manner that achieves an intracellular and/or therapeutic benefit that is not obtainable with a CW light beam.

One or more of a pulsed light beam's temporal pulse width, temporal pulse shape, duty cycle, repetition rate, and/or pulse irradiance can be independently selected such that no portion of a target tissue is heated to greater than about 60° C., of greater than about 55° C., or greater than about 50° C., or greater than about 45° C.

One or more of a pulsed light beam's temporal pulse width, temporal pulse shape, duty cycle, repetition rate, and pulse irradiance can be independently selected such that no portion of a target tissue is heated to greater than about 30° C. above its baseline temperature, or greater than about 20° C. above its baseline temperature, or greater than about 10° C. above its baseline temperature.

One or more of a pulsed light beam's temporal pulse width, temporal pulse shape, duty cycle, repetition rate, and pulse irradiance can be independently selected such that no portion of a target tissue is heated to greater than about 5° C. above its baseline temperature, or greater than about 3° C. above its baseline temperature, or greater than about 1° C. above its “baseline temperature,” which, as used herein, refers, generally, to the temperature of a target tissue prior to irradiation by a light beam. Pulse light beams that may be suitably employed in the systems and methods disclosed herein can have an average radiant power of from about 1 Watt to about 10 Watts or from about 4 Watts to about 6 Watts.

Depending upon the precise phototherapy regimen contemplated, a pulsed irradiation may provide one or more enhancements to cellular functionality and/or therapeutic efficacy. Pulsed irradiation can, for example, provide higher peak irradiances for shorter times, thereby providing more power to deliver to a target tissue while allowing thermal relaxation of the intervening tissue and blood between pulses thereby reducing extent to which an intervening tissue is heated. The time scale for thermal relaxation is typically in the range of a few milliseconds. For example, a thermal relaxation time constant (e.g., the time for tissue to cool from an elevated temperature to one-half the difference between an elevated temperature and a baseline temperature) of human skin is from about 3 milliseconds to about 10 milliseconds, while the thermal relaxation time constant of a human hair follicles is from about 40 milliseconds to about 100 milliseconds. Thus, previous applications of pulsed light to the body for hair removal have optimized temporal pulse widths of greater than 40 milliseconds with time between pulses of hundreds of milliseconds.

While pulsed light within this time scale advantageously reduces the heating of intervening tissue and blood, it does not, however, exhibit optimal efficacy as compared to other time scales. A target tissue, such as an eye or ocular tissue, can be irradiated with pulsed light having parameters that are not optimized to reduce thermal effects but, instead, are optimized to stimulate, excite, induce, and/or otherwise support one or more intercellular or intracellular biological processes that enhance the survival, regeneration, performance, or viability of cells within the target tissue. Thus, the selected temporal profile can result in temperatures of the irradiated tissue that are higher than those resulting from other temporal profiles yet provide improved therapeutic efficacy as compared to those temporal profiles that maintain a target tissue temperature at or near its baseline temperature.

In other aspects of the present systems and methods, pulsing parameters can be selected to favor the kinetics of a biological processes rather than to optimize a tissue's thermal relaxation. The pulsed light beam can, for example, be selected that has a temporal profile (e.g., peak irradiance per pulse, a temporal pulse width, and a pulse duty cycle) that modulates a membrane potential thereby enhancing, restoring, and/or promoting one or more of survival and/or functionality of an irradiated cell, such as a cell that is associated with an ocular disorder, injury, and/or disease. In these aspects of the present systems and methods, a pulsed light can have a temporal profile that supports one or more intercellular or intracellular biological process that is involved in the survival or regeneration of a cell or tissue, such as a retinal cell or tissue, but is not optimized to achieve the thermal relaxation of the irradiated cell or tissue. In such aspects, the cell and/or tissue survives longer after irradiation as compared to a like cell and/or tissue without irradiation. For example, the light can have a protective effect and/or cause a regenerative process in a target cell or tissue.

A temporal profile (e.g., peak irradiance, temporal pulse width, and duty cycle) can be selected to favor the kinetics of a biological process while maintaining an irradiated cell or portion of a tissue at or below a predetermined temperature. This predetermined temperature can be higher than the optimized temperature that can be achieved with other temporal profiles (e.g., other values of the peak irradiance, temporal pulse width, and duty cycle) that minimize the temperature increase of a neighboring cell and/or surrounding tissue due to the irradiation.

A temporal profile having a peak irradiance of 10 W/cm² and a duty cycle of 20% has a time-averaged irradiance of 2 W/cm². Such a pulsed light beam provides the same number of photons to an irradiated surface as does a continuous-wave (CW) light beam having an irradiance of 2 W/cm². Because of the “dark time” between pulses, the pulsed light beam can, however, yield a lower temperature increase than a CW light beam providing the same number of photons to the irradiated surface.

To minimize the temperature increase of the irradiated portion of a tissue, a temporal pulse width and duty cycle can be selected to allow a significant portion of the heat generated per pulse to dissipate before the next pulse reaches the irradiated portion. Thus, rather than optimizing a light beam's temporal parameters to minimize a temperature increase in a target tissue, a temporal parameter can be selected to effectively correspond to and/or to be sufficiently aligned with the timing of a biomolecular processes that is involved in the absorption of a photon thereby increasing therapeutic efficacy. Rather than having a temporal pulse width on the order of hundreds of microseconds, a temporal pulse width can be employed that is not optimized for thermal relaxation of an irradiated tissue (e.g., milliseconds, tens of milliseconds, hundreds of milliseconds). Since such pulse widths are significantly longer than the thermal relaxation time scale, the resulting temperature increases are larger than those of smaller pulse widths but, because of the heat dissipation the time between the pulses, are less than temperature increases resulting from irradiation with a CW light beam.

Various effects of in vitro irradiation of cells using pulsed light have been described in the literature. Incoherent pulsed radiation at a wavelength of 820 nanometers (pulse repetition frequency of 10 Hz, pulse width of 20 milliseconds, dark period between pulses of 80 milliseconds, and duty factor (pulse duration to pulse period ratio) of 20%) on in vitro cellular adhesion has been shown to promote cell-matrix attachment. Karu et al., Lasers in Surgery and Medicine 29:274-281 (2001), which is incorporated in its entirety by reference herein. The modulation of monovalent ion fluxes through a plasma membrane, and not the release of arachidonic acid, was hypothesized to be involved in cellular signaling pathways activated by irradiation at 820 nanometers.

Light-induced changes to the membrane conductance of ventral photoreceptor cells has been found to be dependent upon pulse parameters suggest that two or more processes are involved in light-induced membrane functionalities. Lisman et al., J. Gen. Physiology 58:544-561 (1971), which is incorporated in its entirety by reference herein. Laser-activated electron injection into oxidized cytochrome c oxidase yielded kinetics that establish a reaction sequence of a proton pump mechanism and some of its thermodynamic properties exhibit time constants on the order of a few milliseconds. Belevich et al., Proc. Nat'l Acad. Sci. U.S.A. 104:2685-2690 (2007) and Belevich et al., Nature 440:829-832 (2006), each of which is incorporated in its entirety by reference herein. An in vivo study of neural activation based on pulsed infrared light proposed a photo-thermal effect from transient tissue temperature changes resulting in direct or indirect activation of transmembrane ion channels causing propagation of the action potential. Wells et al., Proc. SPIE 6084:60840X (2006), which is incorporated in its entirety by reference herein.

A temporal profile of a pulsed light beam can include a peak irradiance, a temporal pulse width, a temporal pulse shape, a duty cycle, and a pulse repetition rate or frequency. In those aspects of the presently disclosed systems and methods in which a pulsed light beam is transmitted through a region of a tissues, such as an ocular tissue, at least one of peak irradiance, temporal pulse width, temporal pulse shape, duty cycle, and/or pulse repetition rate or frequency can be selected to provide a time-averaged irradiance (averaged over a time period including a plurality of pulses) at the emission surface of the light source of from about 0.01 mW/cm² to about 1 W/cm², or from about 10 mW/cm² to about 10 W/cm², or from about 100 mW/cm² to about 1000 mW/cm², or from about 500 mW/cm² to about 1 W/cm², or from about 650 mW/cm² to about 750 mW/cm² across the cross-sectional area of the light beam. For example, in certain aspects of these systems and methods, the time-averaged irradiance at a tissue, such as a retinal tissue, is greater than 0.01 mW/cm².

A temporal pulse shape can be generally rectangular, generally triangular, or any one of a wide range of shapes. Pulses can have a rise time (e.g., from 10% of the peak irradiance to 90% of the peak irradiance) of less than 1% of a pulse ON time, or a fall time (e.g., from 90% of the peak irradiance to 10% of the peak irradiance) of less than 1% of a pulse ON time.

Pulses can have a temporal pulse width (e.g., pulse ON time) of from about 0.001 millisecond and about 150 seconds, or from about 0.1 milliseconds to about 150 seconds, or from about 0.01 millisecond to about 10 seconds, or from about 0.1 millisecond to about 1 second, or from about 0.5 millisecond to about 100 milliseconds, or from about 2 milliseconds to about 20 milliseconds, or from about 1 millisecond to about 10 milliseconds. For example, the pulse width can be about 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 milliseconds.

A time between pulses (e.g., pulse OFF time) can be from about 0.01 millisecond to about 150 seconds, or from about 0.1 millisecond to about 100 milliseconds, or from about 4 milliseconds to about 1 second, or from about 8 milliseconds to about 500 milliseconds, or from about 8 milliseconds to about 80 milliseconds, or from about 10 milliseconds to about 200 milliseconds. For example, the time between pulses can be about 4, 8, 10, 20, 50, 100, 200, 500, 700, or 1000 milliseconds.

A pulse duty cycle can be from about 1% to about 80% or from about 10% to about 30%. For example, the pulse duty cycle can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

A peak irradiance per pulse, or pulse energy density, across a cross-sectional area of a light beam at an emission surface of a light source can be from about 0.01 mW/cm² to about 1 W/cm², or from about 10 mW/cm² to about 10 W/cm², or from about 100 mW/cm² to about 1000 mW/cm², or from about 500 mW/cm² to about 1 W/cm², or from about 650 mW/cm² to about 750 mW/cm², or from about 20 mW/cm² to about 20 W/cm², or from about 200 mW/cm² to about 2000 mW/cm², or from about 1 W/cm² to about 2 W/cm², or from about 1300 mW/cm² to about 1500 mW/cm², or from about 1 W/cm² to about 1000 W/cm², or from about 10 W/cm² to about 100 W/cm², or from about 50 W/cm² to about 100 W/cm², or from about 65 W/cm² to about 75 W/cm².

A pulse energy density, or energy density per pulse, can be calculated as the time-averaged power density divided by pulse repetition rate, or frequency. For example, the smallest pulse energy density will occur at the smallest average power density and fastest pulse repetition rate, where the pulse repetition rate is duty cycle divided by the temporal pulse width. The largest pulse energy density will occur at the largest average power density and slowest pulse repetition rate. For example, at a time-averaged power density of 0.01 mW/cm² and a frequency of 100 kHz, the pulse energy density is 0.1 nJ/cm² and at a time-averaged power density of 10 W/cm² and a frequency of 1 Hz, the pulse energy density is 10 J/cm². As another example, at a time-averaged power density of 10 mW/cm² and a frequency of 10 kHz, the pulse energy density is 1 μJ/cm². As yet another example, at a time-averaged power density of 700 mW/cm² and a frequency of 100 Hz, the pulse energy density is 7 mJ/cm².

2. Total Treatment Times

The multi-wavelength phototherapy systems and methods of the present disclosure further provide suitable total treatment times to achieve enhanced cellular functionality and/or improved therapeutic efficacy. A treatment regimen can, for example, proceed continuously for a period of from about 10 seconds to about 2 hours, or from about 1 minute to about 20 minutes, or from about 1 minute to about 5 minutes. For example, the total treatment time can be about two minutes.

In related aspects, the light energy can be delivered for at least one total treatment period of at least about five minutes or for at least one total treatment period of at least ten minutes. The minimum treatment time can be limited by the biological response time (which is on the order of microseconds). The maximum treatment time can be limited by heating and/or to practical treatment times (e.g., completing treatment within about 24 hours of injury).

Light energy can be pulsed during a treatment period or light energy can be continuously applied during the treatment period. If light energy is pulsed, the pulses can be 2 milliseconds long and can occur at a frequency of 100 Hz or can be at least about 10 nanoseconds long and can occur at a frequency of up to about 100 kHz, although shorter or longer pulse widths and/or lower or higher frequencies can be used. For example, the light can be pulsed at a frequency of from about 1 Hz to about 100 Hz, or from about 100 Hz to about 1 kHz, or from about 1 kHz to about 100 kHz, or less than 1 Hz, or greater than 100 kHz.

The treatment may be terminated after one treatment period or the treatment may be repeated for at multiple treatment periods. The time between subsequent treatment periods can be from about five minutes, at least two in a 24-hour period, at least about 1 to 2 days, or at least about one week. The treatment can be repeated multiple times per day and/or multiple times per week. The length of treatment time and frequency of treatment periods can depend on several factors, including the functional recovery of a cell, tissue, and/or patient; the results of imaging analysis of the damaged and/or injured tissue; the disease or condition being treated; the use of pulsed or continuous light; the irradiance of the light; the number of light sources used; and/or the sequence or pattern of the treatment.

Total treatment time can be controlled by the programmable controller. The real time clock and the timers of the programmable controller can be used to control the timing of a particular therapeutic regimen and to allow for scheduled treatment (such as daily, twice a day, or every other day). Timing parameters can be adjusted in response to a feedback signal from a sensor or other device (e.g., biomedical sensor, magnetic resonance imaging device) monitoring the subject.

3. Light Sources

The multi-wavelength phototherapy systems and methods of the present disclosure employ one or more light sources to achieve the delivery of two or more doses of light each dose having a distinct wavelength or range of wavelengths. Suitable light sources include, for example, the office-based, wearable, and/or implantable devices that are described in co-pending PCT Patent Application No. PCT/US15/49261 (Attorney Docket No. LUMI-11-0303WO01; “MULTI-WAVELENGTH PHOTOTHERAPY DEVICES, SYSTEMS, AND METHODS FOR THE NON-INVASIVE TREATMENT OF DAMAGED OR DISEASED TISSUE”), which was filed on Sep. 9, 2015, and U.S. Provisional Patent Application No. 62/048,182 (Attorney Docket No. LUMI-01-0101USP1; “DEVICES AND METHODS FOR NON-INVASIVE MULTI-WAVELENGTH LOW LEVEL LIGHT THERAPY FOR OCULAR TREATMENTS”) and 62/048,187 (Attorney Docket No. LUMI-01-0201USP1; “WEARABLE DEVICES AND METHODS FOR MULTI-WAVELENGTH LOW LEVEL LIGHT THERAPY FOR OCULAR TREATMENTS”), each of which was filed on Sep. 9, 2014. PCT Patent Application No. PCT/US15/49261 and U.S. Provisional Patent Application Nos. 62/048,182 and 62/048,187 are incorporated herein by reference in their entirety.

Other suitable light sources that can be adapted for use in the presently disclosed multiwavelength phototherapy systems and methods include the Warp10™ (Quantum Devices, Inc.; Barneveld, Wis.) and the GentleWaves® (Light Bioscience LLC; Virginia Beach, Va.) instruments. Such light sources can be configured to deliver to a target tissue, such as an ocular tissue, two or more doses of a therapeutically effective amount of low level light having a combination of two or more distinct wavelengths or rages of wavelength.

Such devices for independently delivering, in a targeted fashion, multi-wavelength combinations of low level light to damaged or diseased tissue can be used in combination with sensors and/or other imaging modalities to establish the optimal spatial and tissue parameters to provide an efficacious treatment to the target tissue.

Light sources can be used in the presently disclosed systems and methods in combination with one or more non-light energy sources, such as a magnetic energy source, a radio frequency source, a DC electric field source, an ultrasonic energy source, a microwave energy source, a mechanical energy source, an electromagnetic energy source, and the like.

For example, phototherapy can be combined with OCT, PET, MRI, femtosensors, etc. to provide instruments having therapeutic, diagnostic, tracking, and/or enhanced targeting capabilities for the use of optimizing phototherapy. The light source can optionally include a lens, a diffuser, a waveguides, and/or other optical element or elements.

One or more light emitting diodes (LED) and/or one or more laser diodes can be used as light sources. Laser diodes can be gallium-aluminum-arsenic (GaAlAs) laser diodes, Aluminium gallium indium phosphide (AlGaInP) laser diodes, diode-pumped solid state (DPSS) lasers, and/or vertical cavity surface-emitting laser (VCSEL) diodes. In those applications of the present systems and methods in which multiple light sources are used, the light sources can be coupled to one or more optical fibers. Other light sources that generate or emit light with an appropriate wavelength and irradiance and/or a combination of multiple types of light sources can be used.

The irradiance of the light beam can be selected to provide a predetermined irradiance at a target tissue, such as an ocular tissue. The target tissue, such as an ocular tissue, may be affected by disease or damaged by trauma that has been identified using standard medical imaging techniques. The target tissue, such as an ocular tissue, may be a portion of a tissue that is known to be affected by a particular disease or disorder. For example, the target tissue may be from a portion of an eye that is known to control certain functions and/or processes.

The selection of an appropriate irradiance of a light beam emitted from an emission surface to achieve a desired irradiance at the level of a target tissue, such as an ocular tissue, can include the wavelength of light selected; the nature of the cell and/or tissue being treated; the type of disease, trauma, and/or disorder being treated; the clinical condition of the patient; and the distance between the light source and to the target cell and/or tissue region to be treated.

In some embodiments with a plurality of light sources, certain light sources emit light at a higher or lower power as compared to other light sources. Power output of the light source can thus be tailored depending on the thickness of an intervening tissue, such as an eyelid or cornea, that is between the emission surface of the light source and the target tissue.

LLLT therapy (670 nm) has been implicated in changing the gene expression pattern for multiple genes involved in cellular metabolism (Masha, 2012). Up regulation of several genes involved in electron chain transport, energy metabolism and oxidative phosphorylation were seen, thus rejuvenating the cells metabolic capacity and stimulating the increase in ATP production, which drives other pleiotropic processes, all leading to long-term improvement or normalization of cellular functions. It has been established that phototherapy may affect NFkβ, a major cellular regulator of inflammatory pathways and gene expression. It is not obvious as to the combined benefits of photons from one or more wavelengths to target and regulate gene expression of specific pathways, but the current invention teaches the use of gene expression mapping in multi-wavelength phototherapy to identify characteristics suitable for photobiomodulation applications, which are distinct from those of light used in other applications.

In another embodiment, the use of phototherapy in combination with gene therapy may provide a unique approach to stimulate, enhance or control the regulation and expression of novel genes incorporated into the nucleus through viral vectors or other gene therapy techniques. This is distinct from using light-activated gene products, but to utilize selected wavelengths to naturally stimulate cellular gene expression profiles for newly implanted gene therapy approaches. In a further embodiment, the use of gene therapy has been considered in the regeneration of retinal tissue or to provide for gene therapy in the mitochondrial ocular disorders, such at Leber's hereditary optic neuropathy or AMD. In those cases, gene therapy in combination with LLLT to stimulate specific mitochondrial electron transport protein expression may be contemplated as a better or optimized therapeutic combination approach.

Separately, RNA and protein expression patterns are used by cells to effectively regulate numerous pathways and subsequent cellular activity. The use of multiple wavelengths of light would provide a unique approach to indirectly regulate and improve RNA and protein expression and restore cellular function in damage or diseased tissue. It is unknown as to the individual benefits of photons from one or more wavelengths to regulate protein expression of a specific pathway, but the current invention teaches the use of protein mapping in combination with phototherapy to identify characteristics suitable for photobiomodulation applications, which are distinct from those of light used in other applications. AMD is considered a chronic inflammatory disease wherein protein deposits further propagate the inflammatory state and disease progression. Therefore, the use of multi-wavelength LLLT would have the potential to deliver a unique combination therapeutic, where in the individual wavelengths do not provide for such a therapy. In RPE cell studies, the use of 590 nm light has been shown to inhibit VEGF expression and thus the use of 590 nm LLLT would be useful in one aspect of the treatment of wet AMD subtype. VEGF antibody treatment (Lucentis®) is a currently approved pharmaceutical treatment for wet AMD. Separately, the use of 810 nm LLLT has been shown to improve mitochondrial function, reduce inflammatory markers and prevent β-amyloid deposits in age-related Alzheimer's mice (De Taboada et al, 2011) and could be used in another aspect of the disease. Further, the use of 670 nm LLLT has been shown to reduce inflammatory markers like complement C3 expression and deposition in AMD mouse models but does not affect β-amyloid deposition. Both deposition of lipofusion and β-amyloid have been implicated in the etiology of the diseased eyes in AMD patients. The combinations of multi-wavelengths LLLT alone or the use of multi-wavelengths with an anti-VEGF MoaB, (e.g., Lucentis®, Avastin®), an anti-amyloid drug (e.g., β-secretase inhibitors), an anti-inflammatory drug (e.g., non-steroidal, anti-inflammatory agents, anti-complement agent (e.g., Properidin, C3, MASP-2, C5 inhibitors), antioxidants or vitamin supplements (e.g., AREDS supplements (Lipotriad Visonary™, Viteyes 2®, ICaps®, and PreserVision®, contain similar constituents but either in different proportions, or with additional ingredients) or visual cycle disruptor (e.g., isomerase inhibitors (ACU-4429). These examples provide unique LLLT therapeutic combinations which could represent one or more wavelengths with a device or pharmaceutical or two or more wavelengths of light alone.

In another embodiment, the targeted use of phototherapy to improve mitochondrial function via increased CCO activity, restoration of MMP and regulation of ATP synthesis may be best achieved by the use of multiple wavelengths of light to create the appropriate local cellular response to damage or disease.

Localized cellular conditions in trauma and disease may differ across discrete tissue or organ areas and are under dynamic local regulation. For example, multi-wavelength phototherapy of local CCO activity can lead to release of inhibitory NO from the O₂ binding site. NO is a powerful vasodilator and signal transducer that can regulate the local blood flow to targeted tissue. Thus, the presently disclosed methods may be used to reverse local ischemia or restricted blood flow to damaged or diseased tissue.

The multi-wavelength phototherapy systems and methods disclosed herein provide for the discrete targeting of phototherapy to tissues, such as a retina, and associated surrounding tissue types. As an example, the present multi-wavelength phototherapy systems and methods may be adapted for the treatment of discrete local optic nerve ischemia as seen in non-arteric ischemic optic neuropathy (NAION) or to target anatomical islands of cellular deposits that may be a nidus for inflammation, ischemia, or disease in dry AMD.

In early stage AMD, discrete cellular deposits of lipofuscin can be identified on the retina by standard imaging techniques (OCT, fluorescence imaging). In such a case, the present systems and methods may employ one or more imaging modalities, such as OCT or fluorescence, to facilitate the targeting of the multi-wavelength phototherapy to slow the disease, stop or reverse the deposition of proteins such as lipofuscin or β-amyloid, and/or to reduce, slow or stop the progression of the disease. These aspects of the presently disclosed targeted phototherapy systems and methods provide a disease-modifying approach to chronic ocular disease.

The present multi-wavelength phototherapy systems and methods can, therefore, be employed alone to deliver therapeutically-effective doses of light or may be used in further combination with OCT or other imaging devices (e.g., PET, MRI, Ultra-sound, Doppler, Fluroescence, Femtosensors, etc.) to identify discrete areas of interest thereby facilitating the targeting of cell or tissue boundaries with a combination of wavelengths of light to, thereby, optimize or personalize patient treatment.

Imaging modalities, such as femtosensors, may also be used in combination with the present multi-wavelength phototherapy systems and methods to monitor local retinal O₂ levels to identify AMD patients with local hypoxia to improve treatments and to monitor increased O₂ levels to restore mitochondrial retinal function. It will be understood that the selection of wavelengths, doses, and other treatment parameters may vary depending upon the underlying disorder or disease. The coordinated targeting of multiple wavelengths of light permits the individualized treatment of a patient to restore cellular performance and slow or stop disease propagation. Thus, certain aspects of the present systems and methods can be done alone, in combination with one or more diagnostic devices, and/or with instruments that combine both phototherapy and diagnostic modalities.

In further aspects of the present systems and methods, the desired phototherapy regimen can include selecting appropriate wavelengths and dosing parameters to achieve a desired therapeutic benefit. It will be understood that distinct wavelengths of light exhibit tissue-specific absorption properties, which impact depth of light penetration and, therefore, influence the appropriate dose that is required to achieve therapeutic efficacy.

Additional instrument functionalities, such cameras or other sensors can be employed in the present systems and methods to capture patient-specific features, including orbital features, such as depth, size, skin color, and distances, which permits the dose for each wavelength to be established separately or in combination to optimize treatment parameters. Sensors may also be used to aid in dose selection through the open or closed eyelid, thereby accommodating variations in tissue color and thickness.

In certain systems and methods, including systems and methods for the treatment of a chronic disorder or disease, such as a chronic neurological or ocular condition, a patient may be required to undergo repeated, frequent (e.g., daily) doses of phototherapy. Thus, minimally-invasive phototherapy systems and methods may be employed. For example, in systems and methods for the treatment of intraocular pressure in patients with glaucoma, daily or constant monitoring and phototherapy treatments may be performed at regular intervals. In another example, patients suffering from optic nerve disorders may have limited capacity to institute treatment or may not be physically able to administer treatment. In such instances, minimally-invasive systems and methods of phototherapy may employ an indwelling apparatus, such as an LED.

In some instances, certain parameters of the delivered light may be employed to prevent the scattering by and/or heating of an intervening tissue that is between a light source and a target tissue to which light is delivered. Such parameters that may be varied include the wavelength and/or irradiance of the delivered light. In such instances, light may, for example, be delivered at a low, yet efficacious, irradiances of from about 100 μW/cm² to about 10 W/cm² at a target tissue site. The temporal profile of the delivered light, such as the temporal pulse width, temporal pulse shape, duty cycle, and/or pulse frequency as well as the time period over which the light is delivered can be limited to hundreds of microseconds to minutes thereby achieving an efficacious energy density at the target tissue site being treated.

The target area of a targeted tissue, such as, for example, an optic nerve and surrounding ocular tissue, can include an area of damage, which is referred to herein as a “zone of danger.” The target area of a target tissue can also include a portion of a tissue, such as an ocular tissue, that is outside of a zone of danger. Biomedical mechanisms and reactions that are involved in phototherapy are described in Karu, Proc. SPIE 4159:1-17 (2000) and Hamblin et al., Proc. SPIE 6140:614001 (2006), each of which is incorporated in its entirety by reference herein.

The multi-wavelength phototherapy systems and methods disclosed herein may be employed for the treatment of physical trauma, such as, for example, injury resulting from cataract or lens surgery; for the treatment of inflammation or degeneration of a target tissue; to provide cytoprotection to slow or prevent the irreversible degeneration and loss of a target tissue, such as an ocular tissue, following a primary destructive event; to improve target tissue function, to prevent or slow the progression of loss of target tissue function, and/or to regain previously lost target tissue function; to promote the proliferation, migration, and regenerative cellular properties of endogenous progenitor stem cells for use in the treatment of disease.

In the case of ocular tissue, the term “ocular function” refers, generally, to both visual acuity and contrast sensitivity. Diseases or conditions affecting ocular function include, but are not limited to, primary destructive events include disease processes or physical injury or insult, such as age-related macular degeneration, glaucoma, stroke, diabetic retinopathy, retinitis pigmentosa, CRS, NAION, Leber's disease, ocular surgery, uveitis, cerebral ischemia including focal optic nerve ischemia, and physical trauma such as crush or compression injury to ocular tissues, including a crush or compression injury of the optic nerves or retina, or any acute injury or insult producing ocular degeneration.

As used herein, the terms “therapeutic regimen” and “treatment regimen” refer to a protocol and associated procedures used to provide a therapeutic treatment that includes one or more periods during which light is irradiated to one or more ocular target regions. As used herein, the terms “target,” “target area,” and “target region” refer to a particular ocular area, region, location, structure, population, or projection (e.g., within the retina or optic nerve) to be irradiated by light in association with the treatment of a particular type of ocular condition, disease, disorder, or injury. In certain embodiments, the irradiated portion of the eye can comprise the entire eye. In other embodiments, the irradiated portion of the eye can comprise a targeted region of the eye, such as the retinal region, the macula, or the cornea.

The present multi-wavelength phototherapy systems and methods can be advantageously employed to promote the proliferation, migration, and regenerative cellular properties of endogenous progenitor retinal stem cells for use in retinal or ocular diseases. Stem cells have the capacity to both self-renew and generate postmitotic cells. The retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. It begins as a plastic tissue, capable, in some species, of generating lens and retina, but differentiates early in development and remains normally nonproliferative throughout life. Subpopulations of adult human RPE cells can be activated in vitro to a self-renewing cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers, proliferates extensively, and can redifferentiate into stable cobblestone RPE monolayers. RPESCs are multipotent and, under defined conditions, can generate both neural and mesenchymal progeny, which may be used in replacement therapies and disease modeling.

The present multi-wavelength phototherapy systems and methods can also be advantageously employed to promote the proliferation, migration, and regenerative cellular properties following implantation of retinal stem cells for the treatment of retinal or ocular diseases, such as retinal degenerative disease, which treatment regimen have, historically, been hampered by the limited ability of retinal stem cells to migrate and integrate into a host retina.

The present multi-wavelength phototherapy systems and methods can also be advantageously employed in in vitro methods for preparing cell lysates and membrane enriched and soluble cell fractions thereof, from mesenchymal stem cells and/or ectodermal stem cells.

4. Combination Therapies Including Multi-Wavelength Phototherapy

Within certain embodiments, the present disclosure provides multi-wavelength phototherapy systems and methods that further include the administration and/or delivery to a human patient of one or more small molecule pharmaceutical drug, biologic molecule, or other suitable device to optimize and personalize a given phototherapy treatment regimen for a target tissue, such as an ocular tissue. Within other embodiments, the presently disclosed multi-wavelength phototherapy systems and methods may further include diagnosis and/or monitoring of target tissue damage and/or disease.

The presently disclosed multi-wavelength phototherapy systems and methods can be adapted for the treatment of AMD, which is a chronic inflammatory disease characterized by the formation of protein deposits that propagate an inflammatory state and promote disease progression. Within certain aspects, such multi-wavelength phototherapy systems and methods for the treatment of AMD can include the delivery of a combination of light doses such as, for example, a 590 nm light dose to inhibit VEGF expression; a 810 nm light dose to improve mitochondrial function, reduce inflammatory markers, and to prevent β-amyloid deposits; and a 670 nm light dose to reduce the production and deposition of inflammatory markers such as complement C3, and lipofuscin.

These multi-wavelength phototherapy systems and methods can be used in further combination with one or more anti-VEGF antibodies (e.g., Lucentis®, Avastin®); one or more anti-inflammatory drugs (e.g., non-steroidal, anti-inflammatory agents); one or more anti-amyloid drug (e.g., β-secretase inhibitors); one or more anti-complement agents (e.g., Properidin, C3, MASP-2, C5 inhibitors); one or more antioxidants or vitamin supplements (e.g., AREDS supplements such as Lipotriad Visonary™, Viteyes ICaps®, and PreserVision®); and/or one or more visual cycle disruptors (e.g., isomerase inhibitors (ACU-4429)).

The present disclosure contemplates the use of phototherapy in combination with compositions and methods applicable to cell-based or regenerative therapy for retinal diseases and disorders. In particular, the present disclosure provides multi-wavelength phototherapy systems and methods that further comprise the administration of one or more compositions or devices or that are used in combination with one or more methods for the regeneration or repair of retinal tissue using stem cells (e.g., Very Small Embryonic-like Stem cells (VSELs), mesenchymal stem cells, ectodermal stem cells, etc.). One aspect of the disclosure is a method for treating a retinal disorder with the present phototherapy systems and methods after administering to a patient in need thereof an ectodermal stem cell population to the patient's retinal tissue, and intravenously administering to the patient a mesenchymal stem cell population. The ectodermal stem cells may be derived from fetal neural tissue.

Another aspect of the present disclosure concerns deriving the mesenchymal stem cell population from a source selected from at least one of umbilical cord blood, adult bone marrow and placenta. In still another aspect of the disclosure, the retinal disorder is one or more but not limited to macular degeneration, retinitis pigmentosa, diabetic retinopathy, glaucoma or limbal epithelial cell deficiency.

In certain embodiments, the cells can be induced in vitro to differentiate into a neural or epithelial lineage cells prior to administration and preconditioned with phototherapy. In other embodiments, the cells are administered with at least one other agent, such as a drug for ocular therapy, or another beneficial adjunctive agent such as an anti-inflammatory agent, anti-apoptotic agents, antioxidants or growth factors. In these embodiments, phototherapy treatment can be administered simultaneously with, or before, or after, the postpartum cells. The use of phototherapy systems and methods may be used stimulate the regenerative aspects of the stem cells or use to supplement beneficial adjunctive therapeutic agents or both.

While various embodiments have been disclosed herein, other embodiments will be apparent to those skilled in the art. The various embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the claims.

EXAMPLES Example 1 Treatment of Dry Age Related Macular Degeneration with Photobiomodulation

This Example demonstrates that photobiomodulation (PBM) can be employed advantageously in methods for improving the vision and contrast sensitivity of patients afflicted with dry age-related macular degeneration (AMD).

The study that is presented in this Example was designed as a prospective IRB-approved study in which low powered light at near infrared (NIR), far red and yellow wavelengths was applied, in serial consecutive treatments, to the eyes of patients with dry AMD. Included in this study were patients with dry AMD who were 50 years or older and having best corrected visual acuity (BCVA) ranging from 20/20 to 20/200. Primary outcome measures included: (i) visual acuity, (ii) contrast sensitivity, and (iii) fixation stability. Excluded from the study were subjects with previous or active wet AMD, with a previous history of epilepsy, with cognitive impairment, other retinal disease, previous retinal surgery, significant media opacity, or contraindications to dilation drops.

The absence of neovascularization was ascertained prior to enrollment by examination with Ocular Coherence Tomography (OCT) and Intravenous Fluorescein Angiography (IVFA) and confirmed by a retina specialist.

All subjects were assessed for Visual Acuity with ETDRS charts at 4 meter distance (Precision Vision, USA) recorded in log MAR units, contrast sensitivity at 1.5 and 3 cycles per degree (Stereo Vision Optec 6500, USA) recorded as log contrast sensitivity and for fixation stability with the Nidek MP1 micro perimeter (Nidek Technologies, Padova, Italy). Accurate estimates of fixation stability could be obtained from raw data by calculation of a bi-curve ellipse area (BCEA) as described in Tarita et al., Retina 28:125-133 (2008). Calculations were based on the minor and major axes of an ellipse area covering fixational eye movements and took into account two standard deviation measures of each recorded eye movement. The results were expressed in square degrees.

Measurements took place: (i) prior to treatment; (2) immediately following the treatment protocol; (3) at 6 weeks following the treatment protocol; (4) at 4 months following the treatment protocol; (5) at 6 months following the treatment protocol; and (6) at 12 months following the treatment protocol.

The intervention included the use of low level light therapy (PBM) in the yellow, farred and near infrared (NIR) range using low energy delivery with the Warp10 (Quantum Devices) and the Gentlewaves (Light Bioscience) instruments, which are commercially available and approved by the FDA and Health Canada for use in other conditions. The treatment parameters followed for the Warp10 delivery system were 670 nm±15 nm at 50-80 mW/cm², 4-7.68 J/cm2, for 88±8 seconds. The treatment parameters followed for the Gentlewaves delivery system were 590 nm±8 nm at 4 mW/cm², and 790 nm±60 nm at 0.6 mW/cm², for 35 seconds, pulsed at 2.5 Hz (250 milliseconds on, 150 milliseconds off) while delivering 0.1 J/cm²/treatment. All subjects were treated with the two devices used sequentially at each treatment visit for a total of 18 treatments over a six-week period (3 times per week for 6 weeks).

Data analysis was based on descriptive statistics that included frequency distributions, a measure of central tendency (mean), and a measure of dispersion (standard deviation). A statistical comparison of means between populations was made by t-test and repeated measures analysis of variance (repeated measures ANOVA). Differences were considered to be statistically significant at p values of less than 0.05. The study was performed in adherence to the guidelines of the Declaration of Helsinki. The study protocol was approved by an independent Research Ethics Committee (IRB Services, Aurora, Canada). Informed consent was obtained from all participants.

Over a span of 12 months, 18 AMD study eyes (6 males and 12 females) were recruited and treated; aged 61 to 90 years (mean 74.3 years/SD 7.7).

Repeated measures ANOVA for contrast sensitivity (3 cycles/degree) yielded F (4,68)=11.44 with a p-value of less than 0.0001 and repeated measures ANOVA for contrast sensitivity (1.5 cycles/degree) yielded F (4,68)=4.39 with a p-value of less than 0.0032. Average ETDRS BCVA for the AMD group was measured at 0.25 log Mar units before the treatment and at 0.13 log Mar units 12 months after the treatment (p<0.0001). Repeated Measures ANOVA yielded F(4,68)=18.86 with a p-value of less than 0.0001.

The photobiomodulation treatment regimen disclosed in this example revitalized, rejuvenated, and improved the function of compromised retinal cells on the border of the geographic atrophy with an immediate improvement in visual acuity for a period of 6 months or less.

Contrast sensitivity was statistically significantly improved with the presently-disclosed photobiomodulation treatment regimen. Improvement in contrast sensitivity remained at significant levels at 12 months (FIGS. 1-2).

ETDRS visual acuity was statistically significantly improved immediately following the treatment and this clinical improvement remained at statistically significant levels at 12 months although some decline in the ETDRS log MAR score was evident after 4 months (FIG. 3).

Photobiomodulation is extremely well tolerated. No discomfort was reported and individual treatments were easily dispensed in less than 5 minutes per eye. No significant adverse events were noted during the course of the study described in the present example.

Example 2 Photobiomodulation (PBM) Methods for Decreasing Central Retinal Thickness in Dry Age-Related Macular Degeneration (AMD)

This Example demonstrates, through ocular coherence tomography (OCT) measurements, that photobiomodulation (PBM) can be employed advantageously in methods for decreasing central retinal thickness in the eyes of patients afflicted with dry age-related macular degeneration (AMD).

In a separate non-randomized case series, eight (8) patients afflicted with dry age-related macular degeneration (dry AMD) were treated with multi-wavelength phototherapy over three weeks. Clinical endpoints of CS and VA were conducted as in Example 1. In addition, changes in retinal thickness were determined from consecutive spectral domain ocular coherence tomography (SD-OCT) scans before and after treatment. An overall decrease in central retinal thickness was observed in dry AMD patients immediately following treatment with multi-wavelength phototherapy according to the systems and methods disclosed herein. In total, these data support the clinical and anatomical therapeutic efficacy of those multi-wavelength photobiomodulation therapy systems and methods and confirms the application of those systems and methods for the non-invasive treatment of patients with dry AMD. The results are shown in Table 1.

Patients with dry AMD (determined to have no neo-vascular lesions on retinal inspection, fundus photography, OCT assessment, and IVFA (in some)) that underwent PBM therapy were evaluated with SD-OCT, before and after treatment, with the SPECTRALIS SD-OCT system (Heidelberg Engineering, Carlsbad, Calif.), which combines high-speed image acquisition and custom TruTrack technology to actively track the eye during imaging thereby minimizing motion artifact, enabling noise reduction, and permitting precise tracking over time. The result is point-to-point anatomical correlation between fundus and OCT scans that enables accurate and repeatable alignment of OCT and fundus images, greater image detail and clarity, and more confident assessment of small changes. By integrating SD-OCT with confocal laser scanning ophthalmoscopy (cSLO), the Heidelberg SPECTRALIS platform permits precise follow-up scan placement.

Volume retinal scans were obtained prior to treatment, immediately following the treatment course, and at subsequent intervals following treatment. The same data collection methods that were used in the study presented in Example 1 were utilized for Visual Acuity with ETDRS charts at 4 meter distance (Precision Vision, USA) recorded in letter score, contrast sensitivity at 3 cycles per degree (Stereo Vision Optec 6500, USA) recorded as log contrast sensitivity. In addition, consecutive testing with the Heidelberg Spectralis SD-OCT was used in this group of patients, which revealed changes in retinal thickness.

Patients were treated three times a week for three weeks for a total of nine sessions in which they received the same total dose of PBM as the study described in Example 1, but with a shorter treatment time to facilitate patient compliance. These sessions included the use of PBM in the yellow and red to near-infrared (NIR) range using low-energy delivery with the Warp10 (Quantum Devices) and the Gentlewaves (Light Bioscience) instruments. The treatment parameters followed for the Warp10 delivery system were 670 nm±15 nm at 50-80 mW/cm², 4-7.68 J/cm², for 88±8 seconds. The treatment parameters followed for the Gentlewaves delivery system were 590 nm±8 nm at 4 mW/cm², 790 nm±60 nm at 0.6 mW/cm² for 35 seconds, pulsed at 2.5 Hz (250 milliseconds on, 150 milliseconds off) while delivering 0.1 J/cm²/treatment. These three wavelengths were pre-selected to stimulate the CuA and CuB moieties of mitochondrial cytochrome C oxidase (CCO) activity and to suppress levels of VEGF protein production. All AMD patients were treated with the two devices used sequentially at each treatment visit and then repeated in the same session.

Following is a brief description of each patient and results from the SD-OCT analyses for each.

Subject 1 was a 55-year old Caucasian female who exhibited a reduction in central retinal thickness of 24 microns immediately following treatment, which further reduced to 27 microns at three months. Subject 1 elected to undergo a further 3-week treatment course. At four months the decrease in retinal thickness was 19 microns. Subject 1 had an initial letter score of 39, which increasing to 47 immediately post treatment and to 46 at three months and to 42 following the second course of treatment.

Subject 2 was a 52-year old Caucasian male who exhibited an increase in letter score from 52 to 57 immediately post treatment and a contrast sensitivity increase from log 1.76 to log 2.06. A subtraction scan showed a change of retinal thickness in a post-treatment scan as compared to the pre-treatment reference scan. The central minimum decreased by 20 microns. On this individual section there was an 18 micron decrease in retinal thickness over the highest point of Patient 2's central druse immediately following the treatment.

Subject 3 was a 68-year old Caucasian female who exhibited a 14 micron reduction in central fovea thickness following the treatment protocol. Her letter score increased from 55 to 58 and the contrast sensitivity increased from log 1.60 to log 1.90.

Subject 4 was an 85-year old Caucasian female who had a pre-treatment letter score of 51, which increased to 55 post-treatment, and at 53 one year out from treatment. The log CS score increased from 1.60 to 1.76. The OCT scan at one year showed a decrease of 18 microns. At the one year stage she elected to undergo another course of treatment and three months post treatment the central retinal thickness had decreased to 25 microns from the reference scan. At six months post second treatment the retinal thickness remained decreased at 19 microns. At one-year post second treatment the retinal thickness showed an 18 micron reduction. Three months following second treatment course (17 months from baseline) showing 25 micron reduction. A subtraction thickness map demonstrated an overall central decrease in retinal thickness 20 months from baseline following two treatment courses.

Subject 4's one-year follow-up OCT scan showed an 18 micron reduction in retinal thickness with improvement in CS and VA. Subject 4's three-month post-second treatment course (i.e., 17 months from baseline) OCT scan showed a 25 micron reduction in retinal thickness.

Subject 5 was an 80-year old Caucasian male who had a letter score of 48 prior to treatment increasing to 53 immediately post treatment and a large gain in log CS from 1.00 to 1.90. Two sections (cuts 12 and 13) of Subject 5's post treatment scan showed a decrease of 45 microns and 22 microns respectively.

Subject 6 was a 67-year old Caucasian male who had an initial letter score of 31 increasing to 36 post-treatment and a 19 micron reduction in retinal thickness. Contrast sensitivity increased from log 1.00 to log 1.18.

Subject 7 was an 86-year old Caucasian female who underwent treatment of both eyes. Subject 7 exhibited an initial letter score of 51 increasing to 54 post treatment, 54 at three months and 57 at six months. Log CS scores were 1.6 initially increasing to 1.9 post treatment, 1.76 at three months and 1.6 at six months. Initial letter score of 41 increasing to 43 and remaining at 43 for all subsequent visits. Log CS started at 1.46, remained at 1.46 post treatment, increased to 1.60 at three months and was 1.46 at six months.

The results presented in Examples 1 and 2, which are presented in FIGS. 1-8 and summarized in Table 1, demonstrated anatomical changes in central retinal thickness with a reduction in the central retinal area especially noted directly over the most diseased retina and no reduction in retinal thickness over the normal areas suggesting that the treatment is specifically reducing the retinal thickness over the diseased retina. Anatomical evidence with the resolution of the SD-OCT scans and ensuring the same retinal locations are scanned for serial measurements is unlikely to be influenced by a placebo effect and represents a significant objective end point that can be used in future clinical trials.

TABLE 1 Drusen Reduction in Patients with Dry AMD Following Photobiomodulation Treatment VISUAL ACUITY CONTRAST DRUSEN (ETDRS SENSITIVITY REDUCTION LETTER (LOG UNIT SUBJECT (μM) INCREASE) INCREASE) 1 24 8 0.3 2 20 5 0.3 3 14 3 0.3 4 18 4 0.16 5 45 5 0.9 6 19 5 0.18 7 (OD) 13 4 0.3 7 (OS) 27 2 0.0 MEAN 22.5 4.5 0.305 STANDARD 10.21 1.77 0.26 DEVIATION (SD) STANDARD 3.61 0.63 0.09 ERROR OF THE MEAN (SEM)

The seven subjects who participated in this case series also showed improvements in vision and contrast sensitivity, which were consistent with the improvements that were observed in the dry AMD clinic pilot study that are presented in Example 1. The anatomical evidence presented herein, which resulted from high-resolution SD-OCT scans thus ensuring that the same retinal locations were scanned for serial measurements, represent a significant objective end point, which is unlikely to have been influenced by a placebo effect and can be used in future clinical trials. These objective changes in retinal anatomy following PBM therapy and their correlation with improvement in subjective parameters (i.e., ETDRS, VA, and CS) support the use of PBM for the non-invasive, low-risk treatment of patients with dry AMD.

PBM has been shown recently to cause a significant reduction in focal retinal thickening in non-central diabetic macular edema. Tang et al., Br J Ophthalmol, published online 28Mar14 doi:10.1136/bjophthalmol-2013-304477. While the device used in the study described by Tang et al. was identical to the device utilized in the case series presented in Example 2, the present study with AMD patients employed pulses of yellow and infrared wavelength light that was designed to affect a reduction in vascular endothelial growth factor expression to reduce the conversion of dry AMD to wet AMD. Kiire et al., Retina Today (January/February 2011) (sub-threshold micropulse laser therapy for retinal disorders; Barnstable et al., Prog Ret Eye Res. 23(5):561-577 (2004); Glaser et al., Ophthalmology 94:780-784 (1987); Miller et al., Invest. Ophthalmol. Vis. Sci. 27:1644-1652 (1986); and Ogata et al., Am. J. Ophthalmol. 132(3):427-429 (2001).

Example 3 Further Treatment of Dry Age-Related Macular Degeneration Patients in an Ongoing Patient Data Collection (TORPA II)

The study that is presented in Example 3 was designed as a patient data collection in which low powered light at near infrared (NIR), far red and yellow wavelengths was applied, in serial consecutive treatments, to the eyes of patients with dry AMD. This TORPA II study examined the use of photobiomodulation (PBM) as a treatment for visual outcomes as well as anatomical changes to the retina in subjects with dry AMD. This study included subjects who met the inclusion and exclusion criteria and underwent off-label PBM treatment following conclusion of the previously published TORPA study.

Included in this study were patients with dry AMD who were 50 years or older and having best corrected visual acuity (BCVA) ranging from 20/20 to 20/200. Primary outcome measures included: (i) visual acuity and (ii) contrast sensitivity. Excluded from the study were subjects with previous or active wet AMD, with a previous history of epilepsy, with cognitive impairment, other retinal disease, previous retinal surgery, significant media opacity, or contraindications to dilation drops.

The absence of neovascularization was ascertained prior to enrollment by examination with Ocular Coherence Tomography (OCT) and Intravenous Fluorescein Angiography (IVFA) and confirmed by a retina specialist. All subjects were assessed for Visual Acuity with ETDRS charts at 4 meter distance (Precision Vision, USA) recorded in log MAR units, contrast sensitivity at 1.5, 3 and 6 cycles per degree (Stereo Vision Optec 6500, USA) recorded as log contrast sensitivity. Measurements took place: (i) prior to treatment; (2) immediately following a 3-week treatment protocol; (3) at 3 months following the treatment protocol; (4) at 6 months following the treatment protocol; and (5) at 12 months following the treatment protocol. At this time, patients were still participating in the treatments so a partial listing of the data is presented.

The intervention included the use of PBM in the yellow, farred and near infrared (NIR) range using low energy delivery with the Warp10 (Quantum Devices) and the Gentlewaves (Light Bioscience) instruments, which are commercially available and approved by the FDA and Health Canada for use in other conditions. The treatment parameters followed for the Warp10 delivery system were 670 nm±15 nm at 50-80 mW/cm², 4-7.68 J/cm2, for 88±8 seconds. The treatment parameters followed for the Gentlewaves delivery system were 590 nm±8 nm at 4 mW/cm², and 790 nm±60 nm at 0.6 mW/cm², for 35 seconds, pulsed at 2.5 Hz (250 milliseconds on, 150 milliseconds off) while delivering 0.1 J/cm²/treatment.

The goal of this patient data collection was to reduce the overall number of treatments while maintaining the same total PBM dose and demonstrating safety and efficacy. All subjects were treated with the two devices used sequentially at each treatment visit for a total of 9 treatments over a three-week period (3 times per week for 3 weeks). In the 3-week treatment group, patients were give the same total PBM dose as in the 6-week treatment group, but each session had a double treatment.

Descriptive statistics for all endpoints for each treatment will include the number of subjects, mean, standard deviation, median, minimum and maximum for continuous variables, and frequencies and percentages for categorical variables. Differences were considered to be statistically significant at p values of less than 0.05. Primary analysis.

VA Effect of PBM:

The primary analysis will test the difference in PBM-treated subjects in mean change from BL (pre-treatment) to 3 weeks following treatment in VA. Analysis will use a linear mixed effects model. Exploratory analyses will examine the same endpoint at Months 3 and beyond depending on sample size.

Secondary Analysis. CS Effect of PBM:

The first of the secondary analyses will test the difference in PBM-treated subjects in mean change from BL (pre-treatment) to 3 weeks following treatment in contrast sensitivity. Analysis will use a linear mixed effects model. Exploratory analyses will examine the same endpoint at Months 3 and beyond depending on sample size.

Impact on Retinal Imaging Using Fundus Auto-Fluorescence (FAF) and Optical Coherence Tomography (OCT) of PBM:

The OCT scan will compare reproducible scans at the exact anatomical area of the reference scan and subjects will be scanned at BL for confirmation of dry AMD pathology. Repeat FAF and OCT scans will be taken following treatment and at follow-up visits (e.g., 3, 6 and 12 months). The OCT analysis was exploratory. Descriptive statistics were generated with pre-treatment and post-treatment FAF and OCT scans. For the FAF and OCT imaging analysis, a centralized reader identified anatomic parameters to compare pre- and post-PBM anatomical changes. The OCT analysis examined change from baseline to 3 weeks following treatment. Analysis will use a linear mixed effects model. Exploratory analyses examined the same endpoint at Months 3 and beyond depending on sample size. Drusen volume, mean central 1 mm drusen thickness, and geographic atrophy lesion area after square root transformation were the main outcome measures here. Additionally CRT and retinal volume were assessed. Analyses was performed to compare efficacy in subgroups by AREDS category and by reticular pseudodrusen (RPD) presence or absence. Intact photoreceptor status pre- and post-treatment was compared using a Fisher Exact test. Variables that are not normally distributed may be analyzed using a power transform or using rank values.

Approximately 41 dry AMD study eyes were included in this analysis. The patients were all 3-week treatments. The preliminary data for the 3× per week for 3-week data is shown for VA (FIGS. 5 and 6).

TABLE 2 VA and CS Clinical Improvement and Central Drusen Reduction in Patients with Dry AMD Following Photobiomodulation Treatment Group Mean S.D. (+/−) Visual Acuity @ Baseline (letter score) 41.29 11.36 Visual Acuity @ 3 weeks (letter score) 47.32 11.29 Visual Acuity @ 3 months (letter score) 50.050 6.353 Contrast Sensitivity @ Baseline (log) 1.503 0.229 Contrast Sensitivity @ 3 weeks (log) 1.605 0.243 Contrast Sensitivity @ 3 weeks (log) 1.664 0.181 Central Drusen @ Baseline (volume) 0.460 0.144 Central Drusen @ 3 weeks (volume) 0.445 0.169 Central Drusen @ 3 months (volume) 0.431 0.039

The PBM treatment regimen disclosed in this example also revitalized, rejuvenated, and improved the function of compromised retinal cells on the border of the geographic atrophy with an immediate statistically significant improvement in visual acuity (FIGS. 5 and 6) and contrast sensitivity (data not shown). The clinical benefits were still statistically significant at the 3-month interval for both clinical outcome measures.

Visual acuity was statistically significantly improved immediately following the 3-week treatment and this clinical improvement was similar in benefit to the extended 6-week treatments in the TORPA study (FIGS. 1-13). The two treatments provided the same total dose of the three wavelengths but were optimized to reduce the total number of treatment sessions. Further analysis will determine the frequency of repeated treatments to maintain the maximal benefit.

The data presented in this Example demonstrate that the PBM systems and methods disclosed herein are well-tolerated and, more specifically, that no discomfort was reported, individual treatments were easily dispensed in less than 5 minutes per eye, and no significant adverse events were noted during the course of the presently-described study.

More surprising is that the PBM treatment lead to significant reductions in central drusen volume, the hallmark pathology of the disease. The reduction was evident immediately after the 3-week treatment and was maintained at the 3-month time period following treatment by analyzing the optical coherence tomography (OCT) retinal scans in 19 patients and 33 eyes (Table 3 and FIG. 7). This is the first time that a treatment has shown both clinical as well as anatomical benefits in central drusen volume. No impact was seen on the retinal photoreceptor layer demonstrating the safety of the PBM treatment at the anatomical level with a beneficial reduction in the pathology of dry AMD disease without any local cellular damage. The visual acuity (VA), contrast sensitivity, and central drusen data obtained through the presently disclosed TORPA II study are summarized in Table 2.

TABLE 3 Optical Coherence Tomography Determined Reduction in Central Drusen Volume in Dry AMD Subjects Group Mean p Value Central Drusen Volume .024 P = 0.0008 BL vs V1 (3 week) Central Drusen Volume .029 P = 0.02  BL vs V2 (3 month)

Example 4 Cadaver Studies

Each wavelength has distinct tissue scatter and light penetration properties, thus to ensure the effective delivery of a known intensity of light to ocular tissue, the optical properties of intervening tissues at the wavelengths of interest were obtained through the presently-disclosed study in which light transmission was measured through a set of human cadaver eyes. The design of the experiments performed during and the data obtained from that cadaver study established the retinal fluence rates resulting from the application of light of specific wavelength and power to the ocular region. The results disclosed herein were used to determine the expected retinal fluence rates in the clinical study (TORPA) disclosed in Example 3 and, accordingly, to establish the safety limits for future therapeutic devices.

Power measurements within the eye were taken with an isotropic fiber probe from Medlight (SD200), connected to a silicon-based power detector from Opir (PD300). The detector was connected to an Ophir meter (Nova II). The full device output was measured with a large area, thermal-based power meter from Ophir (L50) 300A, which was connected to the same meter. Spectral measurements were made with an Ocean Optics spectrometer (USB2000) and saved to a laptop. The general procedure for testing each eye is outlined in Table 4, with the measurement locations shown in FIG. 12.

TABLE 4 STEP DESCRIPTION 1 A power out of the device was measured for all three sources. 2 The fiber probe was placed on the eyelid of the cadaver eye and was held in place with tape to the skin on the cheek. 3 The cadaver head was positioned in front of the device, and the device output was adjusted to such that it was centered on the eye. 4 The amber, red, and IR sources were activated sequentially and the power from the probe was recorded for each source. 5 The probe was moved to the cornea, and step 4 was repeated. 6 The spectra from all three sources was measured and recorded. 7 The eyelid was closed over the probe and step 4 was repeated. 8 The spectra from all three sources was measured and recorded. 9 The probe was removed and inserted into the anterior chamber of the eye. The eyelid was opened, and step 4 was repeated. 10 The eyelid was closed and step 4 was repeated. 11 The probe was removed and inserted into the posterior chamber of the eye. The eyelid was opened, and step 4 was repeated. 12 The spectra from all three sources was measured and recorded. 13 The eyelid was closed and step 4 was repeated. 14 The spectra from all three sources was measured and recorded.

For each cadaver, measurements were made on both eyes. Six (twelve eyes) were used in the main study.

Subject data for each specimen in the main study is given in Table 5, along with any applicable observations. Skin color was qualitatively evaluated per the Fitzpatrick skin type classification scale.

TABLE 5 Subject Information Days Post- Skin Subject Gender Mortem Age Race Color Cause of Death Comments 936 F 70 99 Caucasian II Alzheimer's Macular disease degeneration since 2001 949 M 14 72 Caucasian III Cardiovascular Cataract surgery and pulmonary history - unknown collapse date 950 M 21 80 Caucasian III End state renal Status epilepticus disease 957 M 17 83 Caucasian II Respiratory failure, septic shock, pneumonia 956 F 28 86 Caucasian II Dementia 953 F 31 75 Caucasian II Complications MS but it did not from lung cancer affect the visual system

The device output power, P3, for each source was transcribed from the data collection sheets into an electronic database. The raw measurement data were also transcribed from the collection forms. These raw data do not represent true fluence levels, but rather the power delivered from the probe to the Ophir PD300 detector, as indicated on the meter. To convert these to actual fluence rates (mW/cm²), they must be multiplied by a calibration factor to account for the probe collection efficiency, the spectral response of the detector, and isotropy of illumination. Calibration factors were determined for each source though testing and are presented in Table 6.

TABLE 6 Calibration Factors Isotropic Calibration Factors Probe (mW/cm2 per μW) Condition Red Amber IR Wet 9.878 13.949 5.967 Dry 6.315 8.558 3.703 Average 8.096 11.254 4.835

As seen in Table 6, the calibration value for the probe was different depending on whether the probe is wet or dry. Accordingly, the correct value to use was dependent upon the location of the probe during a particular measurement. For measurements made at the eyelid, the “dry” calibration value was used, since the probe was surrounded mostly by air. When taking measurements within the anterior and posterior chambers of the eye, the probe was immersed in fluid, so the “wet” calibration value was used for these readings. The “wet” value was used for measurements at the cornea with the eye closed, since the majority of the probe was in contact with wet tissue at that location. For measurements at the cornea with the open eye, the average value of the wet and dry calibration value was used, since the probe was only in partial contact with wet surfaces during those readings.

These calibration values were applied to the raw recorded data, and the data was then normalized to a common value of 1 W power output from the device for each source. The mean and standard error of each measurement is presented in Table 7. Those data are illustrated graphically in FIG. 13, where the mean fluence rate and standard error are plotted as function of measurement location for the open eye.

TABLE 7 Mean ± SEM of Normalized Fluence Rates for the Open Eye Normalized Fluence Rates (mW/cm²) per 1 W Device Power Mean ± SEM Location Amber Red IR Eyelid 228.10 ± 12.34 169.54 ± 5.64  183.07 ± 4.53 Corneal Surface 249.46 ± 17.86 171.51 ± 6.68  188.25 ± 4.67 (open) Anterior Chamber 176.79 ± 16.94 133.22 ± 11.95  185.77 ± 12.20 (open) Posterior Chamber 15.06 ± 8.27 17.81 ± 5.73 52.90 11.02 (open)

The uncertainty of each measurement is a direct function of the uncertainty of the calibration value. These were calculated for each source and an additional ±3% was added to account for the uncertainty in the meter used to measure the total device power. The resulting total uncertainties were then ±7.3% for the amber, ±14.3% for the red, and ±6.9% for the IR. Uncertainties in the probe location during measurements were estimated to be ±2 mm for the eyelid, cornea, and anterior chamber, and ±5 mm for the posterior chamber. The mean fluence rate and standard error are presented as a function of measurement location for the closed eye in Table 8 and are illustrated graphically in FIG. 14, where the mean fluence rate and standard error are plotted as function of measurement location for the closed eye. The spectra collected at each location were imported into Matlab and normalized for comparison.

TABLE 8 Mean ± SEM of Normalized Fluence Rates for the Closed Eye Normalized Fluence Rates (mW/cm²) per 1 W Device Power Mean ± SEM Location Amber Red IR Eyelid 228.10 ± 12.34 169.54 ± 5.64  183.07 ± 4.53  Corneal Surface 101.21 ± 18.25 120.20 ± 13.89 143.55 ± 18.35 (closed) Anterior Chamber 46.17 ± 9.83  61.70 ± 10.03 107.55 ± 12.56 (closed) Posterior Chamber  4.66 ± 2.19 11.56 ± 3.36 34.98 ± 6.37 (closed)

For each curve, the peak wavelength was determined. The minimum, maximum, mean, and standard deviation of the peak for each source is presented in Table 9.

TABLE 9 Peak Wavelengths Peak Wavelength (nm) Standard Source Mean Min Max Deviation Amber 597.4 594.5 600.1 2.0 Red 668.5 657.9 676.3 7.7 IR 858.1 852.6 861.7 2.9

Some variation in the measured spectra occurred during testing, most notably in the red source. This variation was independent of the measurement location, and was highly dependent on LED power output during the spectral readings. This was understandable and predictable, as there is a dependence of output spectra on LED temperature, which increases as power is increased. Comparing the measured spectra of all the sources to their published specifications shows the range of peak wavelengths all to be within specifications.

For the open and closed eye, the measured fluence rates presented in FIGS. 13 and 14 follow an expected trend, with the highest transmission in the infrared, followed by the red and amber. This is consistent with published transmission data on human tissue.

While the results presented herein are normalized to a device power of 1 W, the fluence rate at the eyelid, prior to transmission through any tissue, is significantly higher with the amber wavelength than in the other two wavelengths. This variation is potentially understood when one considers that the probe is isotropic and collects both the light that is emanating from the instrument, as well as light that is reflected back from the eyelid and surrounding tissue. The variation, then, is indicative of a spectral dependence on the diffuse reflectance of the tissue, with the amber source being reflected at a higher percentage than the red or IR. This is in general agreement with previous measurements of diffuse reflectance of human skin. Murphy et al., J. Biomed. Opt. 10:064020 (2005) and Lim et al., J. Biomed. Opt. 16:011012 (2011).

An additional factor for establishing fluence values at the corneal surface may involve the choice of calibration value at that location. Though the “dry” value was used at the eyelid, the “average” value at the open-eye cornea, and “wet” value at the close-eye cornea, the degree of probe “dryness” or “wetness” in the later two locations could lead to variation. This may result in a more uncertainty on the calculated fluence rates at this location.

The optical emission limits for ophthalmic devices are defined by IEC 15004-2. (International Organization for Standardization. Ophthalmic instruments—Fundamental requirements and test methods—Part 2: Light hazard protection. ISO 15004-2:1-37 (2007)). Emission limits for Group 1 devices, which are defined to produce no emission hazard, are given in Table 10.

TABLE 10 Group 1 irradiance limits per IEC15004-2 Parameter Description Calculation Limit E_(A-R) Retinal photochemical aphakic light hazard $E_{A\text{-}R} = {\sum\limits_{305}^{700}{E_{\lambda} \times {A(\lambda)} \times {\Delta\lambda}}}$ $220\mspace{14mu} \frac{µW}{{cm}^{2}}$ E_(IR-CL) Unweighted coroneal and lenticular infrared radiation irradiance $E_{{IR}\text{-}{CL}} = {\sum\limits_{770}^{2500}{E_{\lambda} \times {\Delta\lambda}}}$ $20\mspace{14mu} \frac{mW}{{cm}^{2}}$ E_(VIR-R) Weighted retinal visible and infrared radiation thermal irradiance $E_{{VIR}\text{-}R} = {\sum\limits_{380}^{1400}{E_{\lambda} \times {R(\lambda)} \times {\Delta\lambda}}}$ $700\mspace{14mu} \frac{mW}{{cm}^{2}}$

Using the above calculations, the specified limits, and the measured device spectra, the maximum irradiance limit for each source at the retina and cornea may be calculated. Results are presented in Table 11. These values assume the device is operating in continuous-wave (CW) mode, and that only a single source is activated at any given time.

TABLE 11 Maximum Irradiance Values per Source, Group 1, IEC15004-2 Location Amber Red IR Retina $110\mspace{14mu} \frac{mW}{{cm}^{2}}$ $220\mspace{14mu} \frac{mW}{{cm}^{2}}$ $1360\mspace{14mu} \frac{mW}{{cm}^{2}}$ Cornea (IR only) NA NA $20\mspace{14mu} \frac{mW}{{cm}^{2}}$

Dividing the values in Table 11 by the normalized fluence rates in Tables 5 and 6 gives the maximum output power of the device such that it may be classified as Group 1 per IEC 15004-2. To be conservative, the values from Tables 7 and 8 used in this calculation are maximums (average+SEM). Results are presented in Table 12. These values provide a direct comparison to commercial instruments and establish the safety class for therapeutic interventions.

TABLE 12 Maximum safe device output, Group 1, IEC15004-2 Maximum Safe CW Device Source Eye State Power (W) Amber Open Eye 4.7 Closed Eye 16.1 Red Open Eye 9.3 Closed Eye 14.7 IR Open Eye 0.10 Closed Eye 0.12

The study presented in this Example provides a definitive understanding of light scatter and penetration in the human cadaver eye and to further establish the safety and efficacy of PBM in the human medical condition. The eye being a unique optical organ requires a special consideration of the light absorption and tissue scatter properties when developing therapeutic applications using light therapy. While cadavers are not living tissue, the anatomical features and tissue properties are very amenable to measurements of tissue scatter and absorption and allow estimates of human exposure of light in the clinical situation. The studies were further undertaken to illustrate the importance of establishing individual wavelength dependent dosing curves that represent actual tissue scatter and absorption for the given wavelength.

The study results presented herein demonstrate measurable light at all levels of the eye with increasing absorption as light penetrated deeper into the eye. Absorption was dependent on the wavelength and the results obtained were consistent with and confirm other studies looking at light scatter and absorption in human tissues. The NIR wavelength of light penetrated most readily in ocular tissue with less loss at the deeper areas including the anterior and posterior chambers. Yellow<far red<NIR were the established order of tissue penetration and supports other multi-wavelength tissue studies. Murphy et al., J. Biomed. Opt. 10:064020 (2005) and Lim et al., J. Biomed. Opt. 16:011012 (2011). These studies are essential to target the retina or other areas of the eye to allow optimization of the clinical doses.

The TORPA study presented in Example 3 (see, also, Merry et al., Association for Research in Vision and Ophthalmology 53:2049 (2012)) utilized two commercial instruments, the Quantum WARP 10 and the Gentlewaves. The wavelengths and doses presented in Table 13 were tested and repeated over a series of 6 weeks.

TABLE 13 TORPA Study Treatment Parameters Irradiance Wavelength at the Eye Duration Pulse Eye Instrument (nm) (mW/cm²) (s) Profile State Warp 10 670 ± 15 50-80 90 CW Closed GentleWaves 590 ± 8  4 30 2.5 Hz, Open 63% DC GentleWaves 790 ± 60 0.6 30 2.5 Hz, Open 63% DC

Based upon the TORPA study parameters and the results obtained in the presently-disclosed cadaver study, the retinal fluence rates presented in Table 14 can be determined for the TORPA study.

TABLE 14 TORPA Study Retinal Fluence Rates, Calculated Wavelength Retinal Fluence Rate Instrument (nm) (mW/cm²) Warp 10 670 ± 15 4.1-6.5 GentleWaves 590 ± 8  0.4 GentleWaves 790 ± 60 0.2

The cellular targets for the three wavelengths are different and thus have independent dose response curves, but the current study provides some context to the tissue exposure that translated to beneficial clinical outcome measures seen in the TORPA study. All fluences are well below the ocular safety standards set by the industry guidelines (i.e., IEC15004). However, there are distinct and surprising differences in the Fluence levels for the three wavelength that make this multi-wavelength approach effective in dry AMD.

The light emitted by the Lumithera LED device is non-coherent, as opposed to the coherent light produced by a laser, and no optical gain occurs within the diode. Consequently, safety standards have evolved to treat LEDs as equivalent to lamps that emit non-coherent light. Applicable standards include those formulated by the International Commission on Non-Ionizing Radiation Protection (ICNIRP), the American Conference of Governmental Industrial Hygienists (ACGIH), and numerous other independent researchers to cover non-coherent light sources. Exposure Limits (ELs) and Threshold Limit Values (TLVs) for LEDs have been established.

Cadaver testing of the three LED sources within the Lumithera device confirmed that the delivered doses of light for the amber, red, and NIR wavelengths were within established safety parameters, provided that the power emitted from the device for each source is kept below the associated value given in Table 14.

In total, the data presented herein support the safety and therapeutic efficacy of multi-wavelength photobiomodulation systems and methods and, moreover, demonstrate an objective improvement in retinal anatomy following multi-wavelength photobiomodulation therapy, which correlates with improved subjective parameters (ETDRS VA and CS). Thus, the data presented herein supports the use of the presently disclosed multi-wavelength phototherapy systems and methods for the treatment of disorders and diseases, including ocular disorders and diseases and, more specifically, dry macular degeneration (dry AMD). 

1. A multi-wavelength phototherapy system for improving or restoring one or more functionality of a target cell or tissue, the system comprising: two or more light sources that deliver light to a target cell or tissue, wherein light from the two or more light sources exhibit distinct wavelength peaks, wherein light from the two or more light sources preferentially stimulates the activity of two or more light sensitive molecules in the target cell or tissue, and wherein the coordinated stimulation of the activity of the two or more light sensitive molecules within the target cell or tissue improves or restores one or more cellular function of the target cell or tissue.
 2. A multi-wavelength phototherapy system stimulating cytochrome c oxidase (CCO) activity in a cell or tissue, the system comprising: a first light source and a second light source that provide coordinated and targeted delivery of light to a target cell or tissue having a CCO with a first light sensitive factor and a second light sensitive factor that are both necessary for CCO activity, wherein light from the first light source exhibits a first wavelength peak that preferentially stimulates the activity of the first light sensitive factor and light from the second light source exhibits a second wavelength peak that preferentially stimulates the activity of the second light sensitive factor, and wherein the coordinated stimulation of the activity of the first and second light sensitive factors by the coordinated and targeted delivery of light from the first and second light sources stimulates the activity of the CCO in the target cell or tissue.
 3. The multi-wavelength phototherapy system of claim 2 wherein stimulation of the CCO activity can improves or restores a target cell or tissue functionality that is associated with a disorder or disease and, thereby, slows or reverses the progression of the disorder or disease.
 4. A multi-wavelength phototherapy system for the treatment of a patient afflicted with a disorder or disease that is associated with a loss or reduction of one or more cellular functionalities, the system comprising: two or more light sources that provide coordinated and targeted delivery of light to a target cell or tissue in the patient, wherein light from each of the two or more light sources exhibits a distinct wavelength peak and wherein coordinating the delivery of light from the two or more sources of light to the target cell restores or enhances a target cell function and, thereby, slows the progression of the disorder or disease.
 5. A multi-wavelength phototherapy system for the treatment of a patient afflicted with an ocular disorder or disease that is associated with an ocular cell exhibiting a lost or reduced functionality, the system comprising: two or more sources of light that provide coordinated and targeted delivery of light exhibiting two or more distinct wavelength peaks to a target cell within an eye of a patient afflicted with an ocular disorder or disease, wherein coordinating and targeting the delivery of light exhibiting the two or more distinct wavelength peaks to an ocular cell restores or enhances the ocular cell functionality and, thereby, slows the progression of the ocular disorder or disease.
 6. A multi-wavelength phototherapy system for the treatment of damaged or diseased ocular tissue in an eye of a human, the system comprising: a. a first light source that delivers to the eye a first therapeutically effective dose of light exhibiting a first wavelength peak and b. a second light source that delivers to the eye a second therapeutically effective dose of light exhibiting a second wavelength peak, wherein the first and the second light sources coordinate and target delivery of the first and second therapeutically effective doses of light to, thereby, reverse, repair, or retard the progression of the damage or disease in the ocular tissue.
 7. The multi-wavelength phototherapy system of claim 6 wherein the system provides simultaneous delivery of the first therapeutically effective dose of light and the second therapeutically effective dose of light.
 8. The multi-wavelength phototherapy system of claim 6 wherein the system provides independent delivery of the first therapeutically effective dose of light and the second therapeutically effective dose of light.
 9. The multi-wavelength phototherapy system of claim 6 wherein each of the light sources is positioned for delivery of light to a target cell within an eye of a patient.
 10. (canceled)
 11. The multi-wavelength phototherapy system of claim 6 wherein one or more of the light sources comprises a light emitting diode, wherein the light emitting diode is optionally a vertical cavity surface-emitting laser diode.
 12. (canceled)
 13. The multi-wavelength phototherapy system of claims 1 or 2, wherein one or more of the light sources are configured to emit a pulsed light beam comprising a plurality of pulses having a temporal pulse width of from 0.1 milliseconds to 150 seconds. 14-18. (canceled)
 19. The multi-wavelength phototherapy system of claim 1 wherein each of the first and the second wavelength peaks is independently selected from 500 nm to 1100 nm.
 20. The multi-wavelength phototherapy system of claim 19 wherein the first wavelength peak or the second wavelength peak is from 570 nm to 600 nm.
 21. The multi-wavelength phototherapy system of claim 19 wherein the first wavelength peak or the second wavelength peak is from 640 nm to 700 nm.
 22. The multi-wavelength phototherapy system of claim 19 wherein the first wavelength peak or the second wavelength peak is from 810 nm to 900 nm.
 23. The multi-wavelength phototherapy system of claim 6 wherein the system delivers light at an irradiance of no greater than 10 W/cm² to an outer surface of an eyelid or a corneal surface of an eye.
 24. The multi-wavelength phototherapy system of claim 23 wherein the system delivers light at an irradiance of from 0.1 mW/cm² to 1 W/cm² at the outer surface of an eyelid or the corneal surface.
 25. The multi-wavelength phototherapy system of claim 23 wherein the system delivers light at an irradiance of from 0.5 mW/cm² to 10 mW/cm² at the outer surface of an eyelid or the corneal surface.
 26. The multi-wavelength phototherapy system of claim 6 wherein the ocular tissue is afflicted with an acute or chronic ocular disorder or disease selected from the group consisting of glaucoma, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, CRS, NAION, Leber's disease, ocular surgery, and uveitis.
 27. The multi-wavelength phototherapy system of claim 6 wherein the ocular tissue is afflicted with an acute or chronic ocular disorder or disease is selected from the group consisting of bleparitis, periorbital wrinkles, seborrhea, and an eyelid skin condition including psoriasis, or eczema.
 28. The multi-wavelength phototherapy system of claim 6 wherein the ocular tissue is afflicted with an acute or chronic ocular disorder or disease, wherein the acute or chronic ocular disorder or disease is an acute injury selected from the group consisting of exposure keratitis, UV keratitis, dry eyes, a viral infection, a bacterial infection, a corneal abrasion, a corneal edema, a surgical incision, a perforating injury, episcleritis, and scleritis.
 29. The multi-wavelength phototherapy system of claim 6 wherein the ocular tissue is afflicted with an acute or chronic ocular disorder or disease, wherein the acute or chronic ocular disorder or disease is an anterior chamber or vitreous disease selected from the group consisting of iritis, vitritis, bacterial endophthalmitis, and sterile endophthalmitis.
 30. The multi-wavelength phototherapy system of claim 6 wherein the ocular tissue is afflicted with an acute or chronic ocular disorder or disease, wherein the acute or chronic ocular disorder or disease is a retinal disease selected from the group consisting of dry AMD, wet AMD, diabetic retinopathy, hypertensive retinopathy, retinitis pigmentosa (RP), a process interfering with a function via a vascular or neurological mechanism, glaucoma, and optic neuritis. 31-61. (canceled) 